Rapid Evolution of Enzymes and Synthetic Micro-organisms for the Development of Industrial Biocatalysts

Lead Research Organisation: University of Manchester
Department Name: Chemistry

Abstract

During the next 10-20 years the chemical industry around the world will undergo a major transformation. As both oil and natural gas begin to run out society will need to look for alternative sources of the chemicals that have traditionally been derived from these fossil fuels. Driven by the need to develop processes that are both economically and environmentally sustainable, the chemical industry will increasingly turn to renewable feedstocks for the manufacture of a growing range of products. Such products are diverse and produced in large volume and include cosmetics, pharmaceutical drugs, components of automobiles and also fuels. This switchover from oil-based starting materials to those derived from biomass will necessitate the development of Industrial (White) Biotechnology-based processes that are able to convert inexpensive raw materials efficiently to high-value products. Industrial Biotechnology involves the use of Nature's catalysts, known as enzymes, for the production of chemicals and related products. The new opportunity that emerges is to be smart with the rational design and construction of engineered biocatalysts and multi-enzyme pathways that are capable of the efficient and robust conversion of simple, low-cost renewable feedstocks (e.g. cellulose, lipids, waste biomass) to each high value end product. In some cases these biocatalytic transformations will be carried out by isolated enzymes, supported on an inert carrier. In other applications, especially for multi-enzyme conversions, the processes will need to be carried out within the environment of a microbial cell - the so-called 'engineered cell factory' - thereby minimizing production costs and rendering conventional manufacturing technologies uncompetitive. The design and engineering of such cells, which are capable of pre-programmed synthetic conversions, represents a significant challenge for IB and will require the interaction of the disciplines of synthetic chemistry, synthetic-systems biology and process engineering.
In collaboration with GlaxoSmithKline (GSK), one of the world's largest pharmaceutical companies, the team of scientists at the University of Manchester will develop a new approach to engineering robust biocatalysts by essentially mimicking the process of Darwinian evolution in the laboratory. This new platform technology will enable us to optimise enzyme for industrial applications in a matter of weeks rather than the months which it currently takes. We will demonstrate the power of this new technology by specially targeting six different synthetic transformations which are on interest to chemists who wish to use biocatalysis in the manufacture of active pharmaceutical ingredients (APIs). These biocatalytic reactions have been specifically chosen because they offer very competitive alternatives to conventional synthetic chemistry methods. These robust biocatalysts will produce the molecules of interest at sufficiently high concentrations, fluxes and yields to ensure economic viability even when crude oil prices are low. At various stages during the project we shall transfer these biocatalysts to GSK who will then apply them to their in-house molecules of interest. We shall also publish the results of the research in the open literature and make the new methods available by web-based tools.

Technical Summary

Industrial Biotechnology is the use of biological resources, such as algae, plants, marine organisms, fungi and micro-organisms, for the production and processing of chemicals, energy and materials. The use of biocatalysts, both as enzymes and also engineered whole cells, in the manufacture of chemicals presents significant advantages in terms of enhanced reaction selectivity, reduced cost of raw materials, lower energy costs, safety and importantly sustainability. The recent explosion in sequence information available from microbial gene sequencing, coupled with a suite of technologies underpinning protein expression, protein engineering, high-throughput screening and bioprocess development, have stimulated chemical companies to consider alternative biotechnology based routes to their products which to date have been produced using traditional chemical feedstocks, reagents and catalysts.
A major limitation at present is the length of time required to develop biocatalysts and biocatalytic processes such that they are fit-for-purpose. From initial identification of enzyme activity, to development of a pilot-scale process, takes at best several months and more likely 1-2 years. This timescale currently restricts the application of biocatalysts mainly to 2nd or 3rd generation manufacturing processes. In this context, recent successes have been achieved in the area of APIs, platform chemicals specialty chemicals and agrochemicals. In order for biocatalysis to be more widely embraced, development times must be reduced to weeks/months thereby bringing this technology in line with existing chemical based technologies. This proposal seeks to develop an accelerated laboratory evolution platform for rapid optimisation of biocatalysts such that they are fit-for-purpose, particularly in the context of industrial application. We shall demonstrate the value of this platform by evolving six different classes of biocatalyst for applications in target molecule synthesis.

Planned Impact

WHO WILL BENEFIT: Our industrial partners GSK are one of the world's largest pharmaceutical companies with a strong track record of bringing new medicines to the market place. Within GSK the newly formed Synthetic Biochemistry Team are committed to developing biotechnology-based processes that are more efficient and sustainable than existing chemical processes that use stoichiometric, metal-based reagents and catalysts and rely upon starting materials largely derived from the oil and petrochemical industry. GSK will therefore benefit in a number of ways by (i) gaining access to the knowledge and methods used for the rapid evolution of biocatalysts and (ii) through the availability of new biocatalyst panels and enzyme libraries, which they can further develop in-house and implement in their expanding portfolio of biotechnological processes. GSK have several manufacturing sites in the UK and elsewhere in the world, where they produce their chemical intermediates and APIs that are formulated in the production of pharmaceuticals and medicines that they sell. These pharmaceuticals are of major benefit to human health and well-being and can contribute to wealth creation and employment in the UK. Access to these biocatalysts, and the underpinning knowledge around methods for their production, will give GSK a competitive edge as they move forward developing low cost and more environmentally friendly manufacturing processes.
HOW WILL THEY BENEFIT: The group at Manchester will work closely with University KT staff and the GSK IP office to secure intellectual property rights for all new inventions we discover. Having secured IP, future development work can take place, and several routes to commercialisation can be explored. For example, new screening methods or genetic algorithms for library design could be protected and the IP could form the basis for a spin-out company or licensing or could be used by GSK as part of their IP protection for in-house molecules of interest to them. The libraries of biocatalysts and their variants we will arising from this research, together with associated plasmids, could be made available to customers via Discovery Biocatalysts which is a non-for-profit company recently established from CoEBio3 within the University of Manchester. Companies purchasing biocatalysts via this route would then be able to screen them against their own target substrates, with a view to licensing specific biocatalytic processes for implementation within their own company. Many pharmaceutical companies (e.g. Merck, AstraZeneca, Pfizer) have their own in-house biocatalysis groups, who may prefer to join in a partnership and to utilise their own in-house expertise to develop a specific biocatalytic process. NJT is the Director of the UK Centre of Excellence in Biocatalysis (CoEBio3), which includes industrial affiliates from ca. 30 pharmaceutical, fine chemical and biotechnology companies who have a specific interest in the industrial applications of enzymes and whole cells. CoEBio3 can therefore open additional pathways for exploiting the research we undertake. Finally, having secured IP, we will actively seek to communicate our scientific findings to the wider research community through scientific meetings and scholarly publications.

Publications

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Description Industrial Biotechnology involves the use of protein catalysts, known as enzymes, for the production of chemicals and related products. The use of these so called biocatalysts in the manufacture of chemicals presents significant advantages in terms of enhanced reaction selectivity, reduced cost of raw materials, lower energy costs, safety and importantly sustainability.

A major limitation at present is the length of time required to develop biocatalysts and biocatalytic processes such that they are fit-for-purpose. From initial identification of enzyme activity, to development of a pilot-scale process, takes at best several months and more likely 1-2 years. An important process that is used to improve biocatalysts is protein engineering where the sequence of amino acid building blocks that make up the enzyme is modified to change the functional properties.

This timescale currently restricts the application of enzymes mainly to 2nd or 3rd generation manufacturing processes. In order for biocatalysis to be more widely embraced, development times must be reduced to weeks/months thereby bringing this technology in line with existing chemical based technologies. The reason why the development timescale is so long is that we lack the tools to accurately predict the function change in enzymes resulting from modifications to the sequence of amino acids. This project seeks to develop an accelerated laboratory evolution platform for rapid optimisation of enzymes such that they are fit-for-purpose, particularly in the context of industrial application.

The work within this project centres on the development of a genetic algorithm that can be used to inform and influence the modifications of enzymes, alterations to the amino acid sequence of a protein that brings about a change in a desired enzymatic activity. A genetic algorithm is a tool which evolves through consecutive rounds of taking inputs to produce results which then form the inputs to the next round. In this case the initial inputs are enzyme sequences which are analysed by the genetic algorithm resulting in suggested modifications to improve the activity. These potentially improved enzymes are then produced in the lab and their activities tested and the results of these tests form the inputs for the genetic algorithm in the next round of evolution. By evolving the genetic algorithm we will develop a powerful tool for predicting how to engineer enzymes for Industrial Biotechnology reducing the time and cost for development.

To demonstrate the power of this new technology we have targeted six different synthetic transformations which are on interest to chemists who wish to use biocatalysis in the manufacture of active pharmaceutical ingredients (APIs). These biocatalytic reactions have been specifically chosen because they offer very competitive alternatives to conventional synthetic chemistry methods.

The team of scientists at Manchester, working in collaboration with GlaxoSmithKline (GSK), are developing a new approach to engineering robust biocatalysts by essentially mimicking the process of Darwinian evolution in the laboratory. This new platform technology will enable us to optimise enzyme for industrial applications in a matter of weeks rather than the months which it currently takes.
Exploitation Route The technology developed within this project will be passed onto the industrial partner who will exploit this for the production of key compounds within their product portfolio. The genetic algorithm will be publicly accessible but this will not be until the completion of the project.
Sectors Agriculture, Food and Drink,Chemicals,Energy,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description All work on the project is currently at too early a stage to understand the impact of the research.
 
Description Future Biomanufacturing Research Hub
Amount £10,284,509 (GBP)
Funding ID EP/S01778X/1 
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Academic/University
Country United Kingdom
Start 03/2019 
End 03/2026
 
Description sLoLa partnership 
Organisation GlaxoSmithKline (GSK)
Department Research and Development GSK
Country United Kingdom 
Sector Private 
PI Contribution We have contributed by developing six biocatalytic platforms for the synthesis of functional molecules for incorporation into advanced pharmaceutical intermediates. We have provided GSK access to the results and material as they are developed to facilitate early adoption by the commercial partner thus maximising the potential impact of the research outcomes.
Collaborator Contribution GSK have provided direct supervision and guidance to the academic research team. this interaction has maintained industrial focus of the research activities.
Impact 10.1002/anie.201311061 10.1002/cctc.201300911 10.1039/C4AN02298J 10.1002/anie.201410670 10.1021/acscatal.5b01132 10.1039/C5SC00913H 10.1126/science.aac9283 10.1021/jacs.5b07326 10.1039/C4OB02282C
Start Year 2012
 
Description Manchester Institute of Biotechnology open day 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact The annual A-Level Open Day was run for the 5th time with the overall aim of raising the profile of biotechnology and its feeder subjects among those considering university and changing the perception of scientists among teenagers and young adults. The day was filled with lab tours, informative talks and interactive demonstrations of various aspects of the research in the institute.

We have received requests for Nuffield Summer Placements of students.
Year(s) Of Engagement Activity 2013,2014,2015,2016
URL http://www.mib.ac.uk/newsandevents/publicengagement/