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

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.

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.

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)