Exploring the biotechnological potential of the UbiD (de)carboxylase family: novel biochemistry for renewable commodity chemicals

The projected depletion of fossil oil resources, and the threat of global warming as a direct consequence of their continued use, has led to the challenge of providing alternative and renewable routes to oil derived fuels and commodity chemicals. However, any such process faces severe demands in terms of yield and costs before it can be considered industrially relevant. In addition, the production of oil derived compounds (ie hydrocarbons) or their functionalization does not belong to what can be considered the "standard" biochemical repertoire. Hence, comparably few biocatalysts, pathways or organisms have been reported to might support such processes. Therefore, while the challenge is clearly defined, the route to a suitable solution if far from clear, and there is an unmet industrial need for new biocatalysts that can produce commodity chemicals such as butadiene (precursor to synthetic rubber) and aromatic dicarboxylic acids (precursors to PET and other polymers). We have recently been able to provide some detailed insights into the mechanism of the UbiD family of enzymes. These act as reversible decarboxylates (ie adding or removing a CO2 group to/from an organic molecule). We and others have shown they are able to interconvert unsaturated hydrocarbons (ie containing a double bond) with the corresponding carboxylic acids (ie version containing the additional CO2 group). A wide range of substrates has been reported, and the reaction catalysed appears readily reversible depending on [CO2] levels. Our recent work on these enzyme has established the achieve this unusual chemistry by making use of a previously unknown enzyme cofactor. In this project, we seek to complete our understanding of how this cofactor is made, and also how it is inactivated, to guide future application.

Building on our fundamental understanding, we seek to derive novel and green production routes to commodity chemicals using renewable feedstocks as UbiD substrates. More specifically, in this grant we will seek to establish to what extend UbiD enzyme can be evolved to produce compounds such as butadiene and aromatic dicarboxylic acids. We will make use of the renewable feedstocks muconic and benzoic acid as UbiD substrates respectively. Our results will provide proof of concept and outline the scope for future biotechnological application.

This application builds upon our recent discovery of a new cofactor essential for activity in the widespread UbiD (de)carboxylase family. In short, we have shown the associated UbiX enzyme prenylates FMN to generate prFMN (prenylated FMN), a cofactor that undergoes oxidative maturation within UbiD. This generates an azomethine ylide form that supports transient 1,3 dipolar cycloaddition with the substrate. The latter step is essential to the reversible decarboxylation carried out by this enzyme family. This raises the tantalising possibility to selectively introduce or remove carboxylic acid groups adjacent to a double bond using UbiD as a biocatalyst. Hence, our proposal seeks to explore and demonstrate the biotechnological scope of the UbiD (de)carboxylases. In short, we seek to derive novel and green production routes to commodity chemicals using renewable feedstocks as UbiD substrates. Our targets consist of butadiene (as a precursor to synthetic rubber) and aromatic dicarboxylic acids (as precursor to PET and other polymers). The renewables muconic and benzoic acid will be used as the respective UbiD substrates. We have preliminary data supporting the fact that Fdc1 (a UbiD family member) can form butadiene, while the related FDCdc can make furan dicarboxylic acids. We will use these enzymes as template for generation of variant libraries, screening for muconic acid depletion (and hence butadiene production) and benzoic acid production (via aromatic dicarboxylic acid decarboxylation) using a XylS/Pm biosensor. Optimised variants will be used to establish proof of concept, after which we will seek suitable follow on funding to ensure industrial application as appropriate.

Beneficiaries:
The outcomes this grant will impact 4 main beneficiaries: (i) The petrochemical industry - new biocatalytic manufacturing processes for generating hydrocarbons such as butadiene (as targeted in this application) mitigate risk associated with the limited supply of reagents from natural resources. 'Natural synthesis' avoids use of toxic/non-renewable reagents with consequent environmental benefits and high acceptability for intermediate and end users. In addition, the tools and systems developed in this application can be further optimised for production of other petrochemical products. (ii) The manufacturing industry - the availability of 'green' products from the petrochemical industry or the ability to use novel green processes to functionalist hydrocarbons (such as carboxylation of benzoic acid as targeted here) will improve the renewable nature and C-footprint of manufacturing processes. In addition, the tools and systems developed in this application will impact widely in emerging biotechnology economies employing 'synthetic biology' to create rationally novel enzymes/pathways used in bulk/fine chemicals manufacture, food production and security. (iii) government policy makers - the generation of novel catalysis to support future synthetic biology applications as outlined in this application is an emerging field that carries significant promise. However, assessing exactly how much scope there is for strategies as outlined in this proposal (to provide alternatives to fossil oil) is urgently needed. The degree of success/scope for translation to other areas will inform both government and industry future funding allocation and strategy. (iv) society in the wider sense - The projected depletion of the oil resources and their ongoing use is a key concern to society. The impact of this event (and the inevitable lead up to it - i.e. unsustainable increase in oil prices) on our society cannot be underestimated. Credible alternatives need to be found, ideally providing "drop-in" replacements for existing petrochemical products so as to be directly compatible with existing infrastructure. This applications seeks to address a small part of this problem (replacing oil derived butadiene/phthalates with a green alternative), but promises to have scope in providing similar answers to a wider range of petrochemicals, ultimately replacing fossil oil.

Exploitation: We anticipate that our newly designed bacterial strains/processes will have commercial impact. Our strategy for translating the technology is to establish IP protection in collaboration with our industrial sponsor and through UMIP (Manchester's IP office). We will communicate through networking events with external stakeholders (industry, other University groups, venture capital groups, policy groups).

Outreach: We anticipate wide interest in the progress and outcomes of this application. We will make use of the internet as the tool with the widest dissemination possibilities, with a range of www based communication tools: websites, podcase and blogs regularly updated and tailored to the various types of audience being targeted (scientific, industrial or general media). In addition, we will ensure direct channels of communication with the applicants and the research staff employed through representation at UK science fairs (Big Bang, Royal Society), engage with local schools and offer summer internships in the applicants' laboratories.