ERA IB 5 A Synthetic Biology approach for bacterial bioconversion of lignin into renewable chemicals (LIGBIO)

Lead Research Organisation: University of Warwick
Department Name: Chemistry

Abstract

Lignin is a polymer found as 15-30% of plant biomass, and is produced industrially as a low-value by-product of pulp/paper manufacture and biofuel production. It contains phenolic monomer units, hence it represents a possible renewable source of aromatic chemicals that are needed industrially to make plastics, pharmaceuticals and agrochemicals, currently derived from crude oil. As part of the effort to develop a sustainable European bio-based chemical industry, we wish to convert lignin into renewable chemicals, however, due to its highly inert structure, no commercial process has yet been developed for conversion of lignin to renewable chemicals. Recently Prof. Bugg's group at Warwick have discovered two novel bacterial lignin-degrading enzymes, and have shown that the lignin breakdown pathways in Rhodococcus jostii RHA1 can be modified genetically in order to generate vanillin, a chemical used in the food/flavour industry. However, at present the metabolic pathways and regulatory systems for lignin breakdown in bacteria are not well understood. Therefore, in this project we will study Pseudomonas putida KT2440, a lignin-degrading bacterium whose genome has been sequenced and whose aromatic metabolic network is the best known in bacteria, to elucidate the metabolic and regulatory networks for lignin degradation. We will then use this knowledge to reprogramme this bacterium, using high activity lignin-degrading enzymes, to produce two target chemicals that are of interest for the production of renewable bioplastics. With industrial partner Biome Bioplastics, we will then develop a fermentation-based process for the production of renewable aromatic chemicals from lignin.

Technical Summary

The major objective of this Project is to capitalise on recent developments in bacterial lignin degradation to rationally engineer efficient biocatalysts for bioconversion of lignin to specialty chemicals of interest for bioplastics manufacture. Lignin is an aromatic heteropolymer that is a component of lignocellulose in plant cell walls, and is produced industrially as a low-value by-product of pulp/paper manufacture and bioethanol production. Bugg has recently shown the feasibility of using gene knockouts in lignin degradation in R. jostii to produce high-value chemicals from lignin. However, at present we have limited knowledge of the metabolic pathways for bacterial lignin degradation, and their regulation, which will be addressed in this Project using lignin-degrading Pseudomonas putida KT2440.

1) We will develop high activity biocatalysts for lignin oxidation. We will identify further members of the bacterial Dyp peroxidase and Mn superoxide dismutase families, and we will use protein engineering and directed evolution to enhance their activity.

2) We will use a systems biology approach to discover the metabolic and regulatory networks for lignin degradation in model organism P. putida KT2440, and develop a metabolic model in this organism.

3) Using synthetic gene cassettes, we will re-route known aromatic degradation pathways in P. putida KT2440 for the production of high value chemicals. We propose two target dicarboxylic acids of interest for synthesis of polyester bioplastics: 5-carboxyvanillic acid, an intermediate on the bacterial biphenyl degradation pathway; and 4-carboxymuconolactone, an intermediate on the beta-ketoadipate pathway.

4) Fermentation-based processes will be developed for the target bio-products in collaboration with the industrial partner, and the bio-products will be converted to polyesters for polymer testing.

Planned Impact

Commercial Impact
The project is jointly with industrial partner Biome Bioplastics Ltd, who are interested in the production of aromatic chemicals from lignin that could be used to make bioplastics. Although the project is for basic/early stage research, it is very likely that outcomes of the project will lead to patentable results and processes. There will be a collaboration agreement drawn up within 3 months of the start of the project to manage intellectual property arising from the project.
Deliverables: Assessment of intellectual property arising from project, leading to patent applications, led by Biome Bioplastics.

Dissemination to schools and general public
The work will also be of interest to the wider public. There is considerable public interest in "green" issues, and the use of biotechnology to convert biomass into renewable chemicals (i.e. useful products from agricultural or even domestic green waste) will be of considerable interest.The results of the project will also be disseminated to the general public and local community through a range of Outreach events, popular science articles, and press releases.
Deliverables:1) Schools outreach events to raise awareness of industrial biotechnology and this specific area of research to local and regional schools; 2) Press releases for specific publications & discoveries during the project (by Biome Bioplastics or University of Warwick)

Publications

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Bugg TD (2015) Enzymatic conversion of lignin into renewable chemicals. in Current opinion in chemical biology

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Rahmanpour R (2017) Identification of an extracellular bacterial flavoenzyme that can prevent re-polymerisation of lignin fragments in Biochemical and Biophysical Research Communications

 
Description We have used protein engineering to enhance the activity of peroxidase Dyp1B from Pseudomonas fluorescens for lignin oxidation. We have also discovered two accessory enzymes for lignin oxidation: dihydrolipoamide dehydrogenase from Thermobifida fusca, and ac acetylesterase enzyme EstK from Pseudomonas putida, and we have studied the activity of a DyP peroxidase enzyme from Thermobifida fusca for lignin oxidation. In collaboration with Dr Eduardo Diaz (CIB, Madrid), we have used transcriptomics to study the genetic basis for lignin oxidation in Pseudomonas putida, and we are currently using this information to engineer Pseudomonas putida for generation of high value chemicals from lignin breakdown.
Exploitation Route The understanding of lignin degradation in a model bacterium, and the development of high activity enzymes for lignin oxidation, could be used to generate high value chemicals from lignin oxidation, which is an important unsolved problem in the generation of a lignocellulose-based biorefinery for production of fuels and chemicals from renewable plant biomass.
Sectors Agriculture, Food and Drink,Chemicals,Energy,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description Collaboration with Biome Bioplastics to generate monomers for bioplastic production from lignin degradation 
Organisation Biome Technologies plc
Country United Kingdom 
Sector Private 
PI Contribution My research group has collaborated with Biome Bioplastics to develop a new process for generation of pyridine-dicarboxylic acids from lignin, by metabolic engineering of lignin degradation pathways in bacteria such as Rhodococcus putida and Pseudomonas putida. In 2015 we published the feasibility of this approach, which was also patented in 2015, and which has led to further collaborative projects.
Collaborator Contribution Biome Bioplastics are interested in making new bio-based plastics from raw materials derived from lignin. They have brought prior knowledge in renewable bioplastics to the collaboration, and they hope to use the outputs of the project to develop a new process for production of a new bioplastic to the market.
Impact Journal publications have been reported elsewhere in Research Fish. A patent application was also made in 2015.
Start Year 2013
 
Description Collaboration with Dr Eduardo Diaz (CIB) to study of the molecular basis for lignin degradation in Pseudomonas putida 
Organisation Centre for Biological Research (CIB)
Country Spain 
Sector Public 
PI Contribution We have collaborated with Dr Eduardo Diaz at CIB-CISC to study the molecular basis for lignin degradation in Pseudomonas putida. We have shared information about potential enzymes and genes likely to be involved in lignin degradation, and we have helped to interpret the transcriptomic data collected. We have also helped to carry out biochemical assays on enzyme candidates identified in his work.
Collaborator Contribution Dr Eduardo Diaz from CIB-CISC has been a partner in the ERA-IB project. His role was to study using transcriptomics the genetic basis for lignin degradation in Pseudomonas putida, and to use this information to engineer Pseudomonas putida to produce high value chemicals from lignin breakdown.
Impact Journal publications arising from the collaboration will be reported via Research Fish as & when they are published.
Start Year 2015
 
Description Collaboration with Dr Stéphanie Baumberger (INRA) for lignin characterisation 
Organisation French National Institute of Agricultural Research
Department INRA Versailles
Country France 
Sector Public 
PI Contribution We have supplied samples of lignin and biologically treated lignin to Dr Baumberger, and discussed the outputs of the lignin analysis with her.
Collaborator Contribution Dr Baumberger has been a partner on the ERA-IB project. Her group has specific expertise in the characterisation of lignin via methods such as thioacidolysis, gel permeation chromatography, and quantitative OH determination via 31P NMR spectroscopy. On the ERA-IB project, her role has been to characterise the changes taking place in the lignin structure as it is degraded by Pseudomonas putida. These methods have also been applied to the study of the mechanism of action of Sphingobacterium sp. T2 manganese superoxide dismutase.
Impact Journal publications will be reported via Research Fish in due course as they are published.
Start Year 2015
 
Description Plenary lecture at Frontiers in Biorefining Conference (Nov 2016, USA) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact 9th November 2016 Plenary lecture at Frontiers in Biorefining (FIB) Conference, 8th-11th Nov 2016, King & Prince Resort,
St. Simons Island, GA, USA. Title: "Bacterial Enzymes for Lignin Degradation: Production of Aromatic Chemicals from Lignocellulose"
Year(s) Of Engagement Activity 2016
 
Description School visit (Abingdon School) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact 19th September 2016 Visited Abingdon School, talked to 6th form students
Talk: "Renewable chemicals from plant biomass: a challenge for the 21st century"
Year(s) Of Engagement Activity 2016
 
Description Seminar at University of Madison-Wisconsin July 2016 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact 19th June 2016 Seminar at Wisconsin Energy Institute, University of Madison-Wisconsin, USA Title: "Bacterial enzymes for lignin degradation"
Year(s) Of Engagement Activity 2016
 
Description Talk at ZELCOR Summer School. 3rd-4th September, Wageningen University, Netherlands "Potential of bacteria and synthetic biology for conversion of lignin" 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Participation in summer school for ZELCOR EU project
Year(s) Of Engagement Activity 2018
 
Description Visit to National Renewable Energy Laboratory (NREL), Colorado, USA 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact 21st July 2016 Seminar at National Renewable Energy Laboratory, Golden, Colorado, USA. Title: "Bacterial enzymes for lignin degradation"
Year(s) Of Engagement Activity 2016