Enzyme co-localisation and aggregation for enhanced metabolic activity for commodity chemicals

Lead Research Organisation: University of Kent
Department Name: Sch of Biosciences

Abstract

Bio-based alternatives to petrochemical commodities, which are required as inputs into a variety of industrial applications such as polymers, coatings and surfactants, are predicted to rise from 2% to 25% of global production over the next ten years. The international chemical market is currently worth around $4 trillion with bio-production offering advantages such as environmental sustainability, cheaper production costs and protection from oil-price volatility. Approaches such as metabolic engineering and synthetic biology can be used to make bio-products and processes more efficient and cost competitive, and are fueling innovation in the chemical industry. In this application we outline a method that offers the potential for a significant step-change in bio-commodity production of a chemical called 1,3-butanediol through the development of intracellular enzyme-aggregates. We will apply this technology to specific bacteria called acetogens that can live on gaseous exhaust fumes.

The project brings together 2 existing and protected technologies (background IP from the University of Kent and ZuvaSyntha) to exemplify the use of enzyme-aggregates in a commercially important strain capable of synthesising commodity chemicals and generate possible novel foreground IP under a joint venture. The use of bacterial microcompartment-derived tags (such as P18 and D18) will be used to provide an intracellular scaffold for the co-localisation of multi-enzyme pathways in microrgansms offering the opportunity to increase flux and catalytic efficiency and protect sensitive intracellular features from the potentially toxic oxyfunctionalised intermediates that arise within the cell. ZuvaSyntha's novel metabolic pathway for the transformation of acetic acid into 1,3-butanediol has been developed for use in anaerobic microorganisms and has been shown by computational metabolic modelling (Prof. David Fell, Oxford Brookes University) to be capable of efficiently converting syngas to BD. By producing the metabolic enzymes with P18 or D18 incorporated into their N-terminii the protein will assemble into an enzyme-aggregate that will represent an internal cellular bioreactor. Such an approach will generate microbial cell factories with significantly enhanced metabolic function.

The results of this project will determine to what extent protein scaffolds and aggregates can be used in acetogens to enhance their metabolic capability for the production of metabolites that are generated via toxic intermediates. The research will reveal the scope to which these various aggregates can be redesigned and re-engineered for the synthesis of important commodities. In so doing the research will provide primary data on the potential that scaffolds have for application in syngas fermentation, through the enhancement of specific metabolic process that are associated with the production of important platform chemicals. The research will also help define an important area of synthetic biology, permitting the exploitation of scaffolds and addressing the basic advantages of internal bioreactors in biology. In this respect, the research will be of general interest to those in applied areas of the biotechnology industry, especially those interested in metabolic engineering. More specifically it will also provide a highly advanced system for the generation of an important commodity chemical in the form of 1,3-butanediol.

Technical Summary

The present proposal couples two separate technologies to produce a novel biocatalyst that contains enzyme-bound scaffolds (using bacterial microcompartment aggregation systems - Technology 1 form Kent) that protect it against product toxicity (i.e. aldehydes) and is able to manufacture high-value commodity chemicals from low value, and potentially polluting, substrates (e.g. acetaldehyde-derived commodity chemicals from acetogenesis - Technology 2 from ZuvaSyntha).

Bio-based alternatives to petrochemical commodities, which are required as inputs into a variety of industrial applications such as polymers, coatings and surfactants, offer advantages such as environmental sustainability, cheaper production costs and protection from oil-price volatility. Approaches such as metabolic engineering and synthetic biology have potential to drive bio-products and processes to be more efficient and cost competitive, and are fueling innovation in the chemical industry. In this application we outline a method that offers the potential for a significant step-change in bio-commodity production through the development of intracellular bioreactors formed by protein aggregation.

Current approaches for enhancing bio-based commodity production are restricted to known biosynthetic pathways and limitations to metabolite toxicity. However, many key bio-commodities are made via aldehyde-intermediates such as acetaldehyde, lactaldehyde and propanaldehyde and their production is often limited because of the inherent toxicity of their chemical reactivity. Ways to reduce this toxicity would offer a significant advantage to the commercial production of these materials. This application outlines a major new strategy to reduce the toxicity of key metabolic intermediates such as acetaldehyde through the deployment of proteinaceous scaffolds.

Publications

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Description We have demonstrated that the use of enzyme aggregates can be used to help improve the yield of commodity chemicals. Specifically, we have shown that enzyme aggregation technology gives a large enhancement for the production of butanediol in E. coli. In essence this project brought together two forms of IP, one held by the University of Kent on enzyme aggregation and the other held by ZuvaSyntha on a novel route for butanediol production involving the enzyme DERA. We have shown that aggregation of the enzymes involved in butanediol production, when aggregated, do indeed result in improved yields of product, demonstrating the proof of concept underpinning this grant works well. This is an important outcome as it is important to develop non-petrochemical ways of generating hydrocarbons, and the development of such procedures is important in helping to reduce emissions and enhancing green chemistry.
Exploitation Route The technology will be of interest to those trying to improve the use of bacteria as cell factories. We are now working to develop strains that can be used for the commercial production of a specific epimer of butanediol. This has been complicated by the fact that ZuvaSyntha are being wound up and we are now looking for alternative partners to help develop this area of science for the industrial biotechnology sector. There are a number of funding schemes available and we are actively speaking with potential investors/backers who are keen to take these ideas forward.
Sectors Chemicals,Education,Manufacturing, including Industrial Biotechology

 
Description We are using this technology to help develop strains for use in producing important commodity chemicals. The project has demonstrated that important bio-commodities can be made via aldehyde-intermediates such as acetaldehyde, despite its inherent toxicity, through improving metabolic flux by providing a higher localised enzyme concentration. The development of aggregates, scaffolds and compartments has helped in the reduction of toxic intermediates and as such therefore offers a significant advantage to the commercial production of aldehyde-derived materials. Our work has shown that pathways improvements through the use of enzyme aggregates has s significant effect on yield. The findings are currently being developed for enhanced production of butanediol, but other commodities such as vanillin are also being targeted with the methodology.
First Year Of Impact 2018
Sector Chemicals,Manufacturing, including Industrial Biotechology
Impact Types Economic