15 NSFBIO - Synthetic Biology for Lignin Utilization

Lead Research Organisation: University of Portsmouth
Department Name: Sch of Biological Sciences

Abstract

Our current reliance on fossil fuels is unsustainable and there is a clear need to find alternative sources of renewable fuels and chemicals to meet the needs of an expanding global population. A vast amount of work has been done in developing the technologies to turn waste plant material (biomass) into sugar that can be fermented to produce bioethanol for our cars, trains and planes. This relies on enzymes, like those used in washing detergents, to break down the plant material so that that it can be converted into biofuels - a direct alternative to fossil fuels. Although the technology now exists to do this, it is simply too expensive. So how can we make it cheaper?

Plant cell walls contain cellulose and it is this polymer that can be broken down into sugars to make fuels. However, up to one-third of plant material is made up of a sticky brown compound called lignin. This is currently vastly underused in commercial plants and it is mainly burned to generate power. This is a huge waste, and one of the reasons that biofuels are currently so expensive. Lignin is actually a very valuable compound, and with the right enzymes, can be turned into useful products such as chemicals, plastics and even carbon-fibre. Not only would these materials be renewable and sustainable, their high value would make biofuels cheaper.

As usual, nature already has the answer. We have found microbes that can live on lignin as a food source and have evolved powerful enzymes break this resistant compound apart. We plan to research these enzymes and link them together in new systems to make them efficient. We will use genetics to help us evolve better enzymes, work out their 3D structures and use advanced computer models to work out the best combinations. If we succeed, we have the potential to make biofuel production commercially viable and create a new range of plant-based products. The result will be transportation fuels that don't rely on dwindling fossil fuel reserves, are sustainable and are kinder to the environment.

Technical Summary

The microbial production of fuels and chemicals from renewable feedstock is a grand challenge for synthetic biology. To date, no microbial chassis has been developed for lignin utilization despite the success of similar approaches with sugars. Although lignin is an abundant, energy-dense polymer that makes up ~30% of plant biomass, it is recalcitrant to degradation. In contrast to cellulose, lignin cannot be readily cleaved into homogenous subunits because it is composed of diverse phenyl-propanoid compounds connected by non-uniform chemical linkages. This complexity makes the targeted degradation of lignin a daunting challenge and results in the dramatic, wasteful underutilization of lignin as a feedstock. In nature, the complete degradation of lignin involves microbial consortia. Although no single organism encodes all the enzymes needed for efficient lignin catabolism, natural metabolic pathways provide a rich catalytic toolbox.

We have assembled a multi-disciplinary team to engineer the first lignin-degrading chassis using a bacterium, Acinetobacter baylyi ADP1. Utilising the unique genetic system of ADP1, we will evolve efficient catabolic devices to expand the degradation of mixtures of lignin-derived aromatic compounds. To complement the microbial genetics, a combination of GC-MS metabalomic and metabolic flux methods will be used to quantify intermediates in the lignin catabolic pathway and in silico metabolic modelling will allow us to target enzymatic bottlenecks and improve lignin catabolism. These specific enzymes will enter our structural biology and protein engineering platform, where we will fully characterise and adapt them with the goal of constructing superior enzyme 'machines' for the efficient conversion of lignin to desired products. These engineered enzymes will then be incorporated back into ADP1 and multiple iterative cycles will allow us to continually improve the efficiency of the system.

Planned Impact

The aim of this research is to produce enzymes with enhanced lignin degrading activities that can be used directly in the production of fine chemicals from waste material to support commercially viable processes. Crucially, this technology uses waste biomass and therefore does not compete with food crops, making it a sustainable alternative to fossil-based transportation fuels and provide a new and sustainable source of high-value chemicals. Our main objectives directly address several BBSRC strategic priority areas:

Synthetic biology:
Adopting an enzyme engineering approach, informed by metabolomics and driven by genetic evolution, to generate enhanced enzymes and novel metabolic pathways.

Bioenergy:
Addressing a key economic bottleneck in 2nd Generation bioethanol production and sustainable fine chemicals.

Industrial biotechnology:
The newly developed recombinant enzymes may find application in other industrial sectors such as paper and textile manufacturing and biological washing detergents. New enzymes have the potential to aid in the manufacture of natural products and drugs that remain difficult to synthesise.

Increased international collaboration:
Capitalising on the world-class infrastructure between the 3 NSF-funded groups in the USA, the DoE-funded facility at NREL and the biophysics and structural biology expertise in the UK.

Public engagement:
For public engagement, previous work in related areas has already proved to be particularly attractive to the media and has headlined national TV news, newspapers and in excess of 20 websites worldwide (Google: 'gribble mcgeehan'). Bodies such as the BBSRC, STFC have also covered this work together with large facilities such as the Diamond Light Source, UK and the National Science Foundation, USA.

Publications

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Wolf ME (2022) Cytochromes P450 in the biocatalytic valorization of lignin. in Current opinion in biotechnology

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Machovina MM (2019) Enabling microbial syringol conversion through structure-guided protein engineering. in Proceedings of the National Academy of Sciences of the United States of America

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Knott BC (2020) Characterization and engineering of a two-enzyme system for plastics depolymerization. in Proceedings of the National Academy of Sciences of the United States of America

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Kincannon WM (2022) Biochemical and structural characterization of an aromatic ring-hydroxylating dioxygenase for terephthalic acid catabolism. in Proceedings of the National Academy of Sciences of the United States of America

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Barham T (2021) Thirty-five years later: Long-term effects of the Matlab maternal and child health/family planning program on older women's well-being. in Proceedings of the National Academy of Sciences of the United States of America

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Austin HP (2018) Characterization and engineering of a plastic-degrading aromatic polyesterase. in Proceedings of the National Academy of Sciences of the United States of America

 
Description We have solved the first 3D structure of a novel enzyme that can breakdown a key component of lignocellulose plant material call guaiaicol.

We have successfully engineered this enzyme to accept a new highly abundant lignocellulose substrate, syringol.

We have also solved and engineered an enzyme that can digest PET plastic and this work was awarded the Times Higher Education STEM Research Project of the year (2019).

On the basis of our recent research successes, we have created a new Centre for Enzyme Innovation (CEI) which has attracted £5.8m in funding from Research England.
Exploitation Route These data will eventually be used to design new synthetic pathways to turn plant waste into useful chemicals that can currently only be generated from fossil-based extraction.

We are now looking at routes to commercialize PET digesting enzymes for circular recycling.
Sectors Chemicals

Energy

Environment

Manufacturing

including Industrial Biotechology

URL https://www.port.ac.uk/news-events-and-blogs/news/portsmouth-scientists-awarded-major-plastics-funding
 
Description Our recent work involved solving the structure and improving the function of an enzyme (PETase) which can break down some of our most commonly polluting plastics. This work captivated a global audience of over 200 million people and features in the UKRI Strategy Prospectus, BBSRC and Diamond Light Source highlights, is cited in international policy papers and reached the Altmetric Top 100 from 2.8 million publications. I am now routinely invited to speak at high profile international conferences, government meetings and in the media (e.g. UK, USA, China) on sustainable enzyme solutions to plastic pollution. Our work on lignin and plastic active enzymes is expanding and converging as the key chemicals in both breakdown processes share similar structure and pathways. Our latest paper on MHETase also reached the Altmetric Top 100 in 2020.
First Year Of Impact 2018
Sector Environment,Manufacturing, including Industrial Biotechology
Impact Types Societal

Policy & public services

 
Description Case study in UKRI Strategic Prospectus
Geographic Reach National 
Policy Influence Type Citation in other policy documents
URL https://www.ukri.org/about-us/strategic-prospectus/creating-social-and-cultural-impact/engineering-a...
 
Description Cited by Analysis & Policy Observatory (APO) - Synthetic biology in Australia: an outlook to 2030
Geographic Reach Asia 
Policy Influence Type Citation in other policy documents
URL https://apo.org.au/node/190361
 
Description Frontières génétiques pour la conservation...évaluation technique
Geographic Reach Multiple continents/international 
Policy Influence Type Citation in other policy documents
Impact Citation of our paper: Characterization and engineering of a plastic-degrading aromatic polyesterase, PNAS 2018
URL https://portals.iucn.org/library/node/48915
 
Description Centre for Enzyme Innovation - Research England - Expanding Excellence in England (E3)
Amount £5,827,978 (GBP)
Funding ID University of Portsmouth Centre for Enzyme Innovation 42.18 
Organisation United Kingdom Research and Innovation 
Department Research England
Sector Public
Country United Kingdom
Start 07/2019 
End 07/2022
 
Description NREL subcontract - New enzymes for the deconstruction of natural and synthetic polymers
Amount $320,000 (USD)
Organisation U.S. Department of Energy 
Sector Public
Country United States
Start 09/2018 
End 09/2022
 
Title X-ray protein structures 
Description Published X-ray structures to date 
Type Of Material Database/Collection of data 
Year Produced 2008 
Provided To Others? Yes  
Impact Multiple publications 
URL http://www.ebi.ac.uk/pdbe/entry/search/index?text:mcgeehan%20je
 
Description Collaboration for advanced enzyme molecular simulations 
Organisation University of South Florida
Country United States 
Sector Academic/University 
PI Contribution We have provided high-resolution X-ray structures.
Collaborator Contribution Extensive ligand docking and molecular dynamics simulations.
Impact Manuscript in submission
Start Year 2017
 
Description Collaboration with enzyme biochemistry expert 
Organisation Montana State University
Country United States 
Sector Academic/University 
PI Contribution Provision of purified enzymes
Collaborator Contribution Detailed biochemical analysis
Impact Manuscript under review
Start Year 2017
 
Description Collaboration with the Eltis Group, University of British Columbia 
Organisation University of British Columbia
Country Canada 
Sector Academic/University 
PI Contribution We have shared data and materials and are now currently working on joint projects stemming from the original work on P450s funded by this grant.
Collaborator Contribution They have supplied DNA clones, protocols and purified enzymes.
Impact We have co-produced several enzyme structure and hope to publish in 2021/22
Start Year 2017
 
Description Collaboration with world-expert in enzyme mechanisms 
Organisation University of California, Los Angeles (UCLA)
Country United States 
Sector Academic/University 
PI Contribution Provided platform of biochemical and X-ray structural data
Collaborator Contribution Enzyme mechanism analysis using advanced density functional theory calculations
Impact Manuscript under review
Start Year 2017
 
Description Diamond Light Source beamline staff collaboration 
Organisation Diamond Light Source
Country United Kingdom 
Sector Private 
PI Contribution Provisions of proteins and crystals
Collaborator Contribution Provision of structural expertise and state-of-the-art X-ray beamline facilities
Impact Several enzyme structures solved, manuscripts in preparation.
Start Year 2014
 
Description Industrial partnership with GlaxoSmithKline for enzyme production 
Organisation GlaxoSmithKline (GSK)
Country Global 
Sector Private 
PI Contribution We are supplying bacterial expression systems for the production of the PETase enzyme
Collaborator Contribution GSK are providing enzyme fermentation scale-up expertise and facilities
Impact Research is ongoing. We were jointly interview by BBC world service about this project.
Start Year 2018
 
Description Synthetic biology working partners in the USA 
Organisation Northwestern University
Department Center for Synthetic Biology
Country United States 
Sector Academic/University 
PI Contribution Shared data and provided funding for a joint proposal to the University of Georgia
Collaborator Contribution Materials including expression plasmids and constructs. Bacterial evolution expertise and genomic sequencing services.
Impact We have developed new strategies for the evolution of lignin degradation enzymes. This is a multi-disciplinary combination of structural biology (UK) and synthetic biology (USA).
Start Year 2016
 
Description Synthetic biology working partners in the USA 
Organisation University of Georgia
Department Department of Microbiology
Country United States 
Sector Academic/University 
PI Contribution Shared data and provided funding for a joint proposal to the University of Georgia
Collaborator Contribution Materials including expression plasmids and constructs. Bacterial evolution expertise and genomic sequencing services.
Impact We have developed new strategies for the evolution of lignin degradation enzymes. This is a multi-disciplinary combination of structural biology (UK) and synthetic biology (USA).
Start Year 2016
 
Description US Department of Energy subcontract 
Organisation U.S. Department of Energy
Department National Renewable Energy Laboratory (NREL)
Country United States 
Sector Public 
PI Contribution Biophysical and structural characterisation of novel lignocellulose-digesting enzymes
Collaborator Contribution Provision of genes and protein expression systems.
Impact A joint 4-year studentship 2015-2019
Start Year 2015
 
Description Almedalen - Swedish Political Retreat Invited to speak by British Embassy 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Invited to speak by British Embassy to Almedalen - Swedish Political Retreat
Dscussion Panel 4 July 2018
Marine plastics: how can researchers, artists and governments work together to develop solutions?
Year(s) Of Engagement Activity 2018
URL http://program.almedalsveckan.info/event/user-view/54648
 
Description BBC Interview 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact Interview with Kate Stephens BBC News, Science Producer and David Shuckman, BBC Broadcasting House, Central London
Year(s) Of Engagement Activity 2018
URL https://www.bbc.co.uk/news/science-environment-43783631
 
Description Blow Moulding Conference 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact Conference talk: Engineering biological enzymes for deconstructing and recycling PET
Year(s) Of Engagement Activity 2019
 
Description British Crystallography Association Plenary 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Talk - Focusing X-rays on plastic pollution
Year(s) Of Engagement Activity 2019
 
Description Cafe Scientifique - Talk IOW 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Public engagement for plastics research
Year(s) Of Engagement Activity 2018
 
Description Café Scientifique Henley 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact How plastic-eating bacteria actually work
Year(s) Of Engagement Activity 2019
 
Description Inaugural public lecture 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact An inaugural public lecture was given to a very mixed audience of around 200, from students, to academics, the general public and our local MP, Flick Drummond. A full video will be online shortly.
Year(s) Of Engagement Activity 2017
URL http://uopnews.port.ac.uk/2017/02/13/free-public-lecture-shines-a-light-into-our-biological-world/
 
Description Innovate UK Plastics CEI Discussions 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Invited delegate for discussion on government funding for plastic recycling projects
Year(s) Of Engagement Activity 2018
 
Description Institute of Mechanical Engineers Meeting 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Professional Practitioners
Results and Impact Falling out of love with the wonder material - open debate with audience and local MPs
Year(s) Of Engagement Activity 2019
 
Description International Public Awareness of Research Infrastructure Plenary @ Diamond Light Source UK 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Science and Society: Engaging with public with plastic-eating enzymes
Year(s) Of Engagement Activity 2019
 
Description International Resource Panel - Marine Plastic Litter Workshop 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Policymakers/politicians
Results and Impact This workshop is a contribution to the delivery of the G20 Osaka Blue Ocean Vision (2019) which aims to "reduce additional pollution by marine plastic litter by 2050 through a comprehensive life-cycle approach that includes reducing the discharge of mismanaged plastic litter by improved waste management and innovative solutions
Year(s) Of Engagement Activity 2020
 
Description Invited delegate House of Commons - Funding Meeting 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Policymakers/politicians
Results and Impact Our research was highlighted in the launch of a new government programme - A Vision for Zero Plastic Waste
Year(s) Of Engagement Activity 2018
URL https://www.policyconnect.org.uk/events/launch-findings-vision-zero-plastic-waste-project
 
Description Keynote Speaker at the BPF 'Future of Chemical Recycling' Webinar 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact This BPF webinar, presented in partnership with PlasticsEurope took place on 30 June 2020. The event provided attendees with an in-depth exploration of Chemical Recycling. The event also examined the current position of Chemical Recycling within the recycling infrastructure as well as investigating what the future will bring.
Year(s) Of Engagement Activity 2020
URL https://www.bpf.co.uk/events/webinars/the-future-of-chemical-recycling.aspx
 
Description Local newspaper highlight on BBSRC projects 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Local interview and press release in local newspaper.
Year(s) Of Engagement Activity 2017
URL http://www.largsandmillportnews.com/news/15099648.Groundbreaking_Largs_professor_in_new_role/
 
Description MIT Technology Review - Hong Kong 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact Invited speak at international technology conference. Industries and investor interest.
Year(s) Of Engagement Activity 2018
URL https://www.emtechhk.com/
 
Description National and International News and Media Interview 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact Following the release of our research paper on a plastic-digesting enzyme in PNAS (https://www.pnas.org/content/115/19/E4350) the story was picked up by many media outlets, going out to a global audience of over 150 million people.

Coverage includes the press (The Times, The Guardian, and The Economist), television media (BBC,ITV,CNN, CBS, Al Jazeera, China Global News Network, and HBO), and by funding organisations, including the BBSRC and UKRI.
Year(s) Of Engagement Activity 2018
URL https://en.wikipedia.org/wiki/John_McGeehan
 
Description Opening speaker - EMTech Singapore 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact EmTech Asia: Emerging Technologies Conference
Year(s) Of Engagement Activity 2019
URL https://emtechasia.com/
 
Description Panel - Arctic Circle Assembly 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Open discussion panel on marine plastics pollution
Year(s) Of Engagement Activity 2018
URL http://www.arcticcircle.org/assemblies/2018
 
Description Panel member for MAXIV, Europes newest synchrotron 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Ongoing contribution to international biological crystallography panel at MAXVI, Lund
Year(s) Of Engagement Activity 2017,2018
 
Description Portsmouth City Council 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Policymakers/politicians
Results and Impact The discovery of bacteria that can digest plastic
Year(s) Of Engagement Activity 2019
 
Description Presentation to Society of Chemical Industry/Royal Society of Chemistry 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Talk - Plastics, from Cradle to Grave
Year(s) Of Engagement Activity 2019
 
Description Presented current research projects to 27 UKRI Executive board members 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Policymakers/politicians
Results and Impact Presented latest research to UKRI Executive board members during their visit to the Diamond Light Source
Year(s) Of Engagement Activity 2018
 
Description Probus Organisation 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Turning the tide on plastic pollution with enzymes
Year(s) Of Engagement Activity 2019
 
Description Public talk - Southampton Boat Show 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Public engagement talk on the effects of plastic pollution and innovative solutions
Year(s) Of Engagement Activity 2018
 
Description Recording of video content for new Diamond VMXi beamline 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact An active member of the VMXi working group at the Diamond light source.
Year(s) Of Engagement Activity 2017
URL https://vimeo.com/203828475
 
Description Roundtable at Lambeth Palace, Archbishop of Canterbury 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Worldwide communities affected by plastic pollution
Year(s) Of Engagement Activity 2019
 
Description Royal visit and press 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Involvement with new beamline opening at the Diamond Light Source as 'first academic users'. This presented an opportunity to engage with HRH Princess on on our research project.
Year(s) Of Engagement Activity 2017
URL http://uopnews.port.ac.uk/2017/02/15/new-beamline-to-boost-advances-in-biomedical-research/
 
Description Rutherford Appleton Laboratory - Talking Science 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Engineering enzymes to digest our most polluting plastics
Year(s) Of Engagement Activity 2020
 
Description Solent Energy & Environment Group, HMS Excellent Naval Base 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Industry/Business
Results and Impact Can new enzymes help tackle plastic pollution?
Year(s) Of Engagement Activity 2019
 
Description Talk and panel - Westminster Energy, Environment & Transport Forum 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Westminster Energy, Environment & Transport Forum: Reducing waste from single-use plastics: innovation, legal issues and incentives
Year(s) Of Engagement Activity 2018
URL https://www.westminsterforumprojects.co.uk/agenda/reducing-plastic-waste-2018-agenda.pdf
 
Description Talk to artists at St Martin's College 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Postgraduate students
Results and Impact Provided a lecture on the Art of Enzymes to postgraduate students: MS Material Futures
CSM - University of the Arts London
Year(s) Of Engagement Activity 2018
 
Description U3A Science and Technology 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Biological solutions for plastic recycling
Year(s) Of Engagement Activity 2020
 
Description • House of Lords - Addressing the Plastic Challenge 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Policymakers/politicians
Results and Impact The Portsmouth Centre for Innovation
Year(s) Of Engagement Activity 2019