Engineered Biofilm Catalysts

Historically, biofilms are predominantly perceived as problematic; associated with infections, dental caries, marine and reactor fouling and reduction in heat transfer. In their natural environment individual planktonic cells have the tendency to cluster together at surfaces and interfaces. Within these clusters they protect themselves from environmental and chemical stress by secreting extracellular polymeric substances (EPS) a protective and adhesive polysaccharide matrix. This EPS gives the biofilm a degree of protection against extremes of pH, temperature or the introduction of organic solvents which cannot be tolerated by individual cells. In the current global climate of searching for increasingly green approaches to the synthesis of fine chemicals and pharmaceuticals there is a growing interest in employing biotransformations: using cells to produce useful products. Although such a strategy enables the generation of enantiomerically pure compounds, the reduction of steps in a synthetic route and the possibility of eliminating environmentally detrimental catalysts, solvents and reagents, their use is problematic due to the fact that individual cells cannot in general tolerate the extreme conditions required. In this research we exploit biofilms to produce highly active Engineered Biofilm Catalysts (EBC). This is a new area as the use of biofilms in biotransformations has been largely confined to wastewater treatment and bioremediation generally by mixed microbial communities referred to as consortia. Reports of the use of biofilms for synthesis of compounds are few and far between, industrially the list of compounds generated in such a manner is little more extensive than simple compounds such as acetic acid, ethanol, butanol, 2,3-butanediol, lactic acid, fumaric acid, and succinic acid. This interdisciplinary proposal presents a novel methodology for the development and exploitation of engineered biofilm catalysts (EBC) for biotransformations relevant to the fine chemicals and pharmaceutical industries. Recently we have demonstrated the generation and utilisation of an Engineered Biofilm Catalyst (EBC). The immobilised EBC not only demonstrated unprecedented stability but proved to be a strikingly better catalyst than the free cells. As the EBC is artificially generated there is considerable scope to engineer its microstructure. We propose to explore the general application of EBC to catalysis as well as to investigate how microstructure and gene expression relate to catalytic activity. We will employ EBC using flow chemistry to generate a prototype EBC reactor for biotransformations as an exemplar for future industrial exploitation.

In the current global climate of searching for increasingly green approaches to the synthesis of fine chemicals and pharmaceuticals there is a growing interest in employing biotransformations. Although such a strategy enables generation of enantiomerically pure compounds, reduction of steps in a synthesis or the possibility of eliminating environmentally detrimental materials, their use is problematic as individual cells cannot in general tolerate the extreme conditions required. This interdisciplinary proposal presents a novel methodology to exploit the tolerance to extreme conditions of biofilms in highly active Engineered Biofilm Catalysts (EBC). This is a new area: the use of biofilms in biotransformations has been largely confined to wastewater treatment and bioremediation generally by consortia. Recently we have demonstrated the generation and utilisation of an EBC using Escherichia coli K-12 ompR234 pretransformed with pSTB7 a plasmid enabling the over expression of tryptophan synthase. This enzyme mediates generation of a series of L-halotryptophans from serine and haloindoles. The immobilised EBC not only demonstrated unprecedented stability but proved to be a strikingly better catalyst than the free cells. In this research we propose to -Investigate physical and chemical parameters governing the microstructure and activity of the K-12 E. coli EBC and make preliminary investigations as to the role of gene expression in specifying the structure and function of the engineered biofilm; -Apply the EBC concept to other biotransformations and exploring the possibility of generating an EBC with other organisms for example antibiotic producers such as Saccharopolyspora erythraea; -Explore the possibility of a multifunctional EBC (in order to produce multiple products) using gene promoter switching; -Provide a preliminary assessment of scalability and feasibility of an EBC bioreactor via construction of a lab-scale prototype for flow chemistry

Through this research we have potential to make a demonstrable contribution to impact over a multiple year timeframe. The main focus of impact will be Scientific Advancement and Knowledge (0-3+ years). The fundamental research programme within this project will be disseminated via high quality scientific journals in the field (e.g. Biotechnology and Bioengineering, Nature Biotechnology, Angewandte Chemie). We intend to present our work at 2 leading international conferences as well as national meetings. Processes and Products (3-10 years). The potential beneficiaries of this research programme beyond the project timescale are industries involved in the manufacture of fine chemicals, pharmaceutical intermediates and pharmaceutical products. Whilst the research is currently fundamental in nature and at a low level of technology readiness, through this proposal we will engage with industry at the earliest opportunity via our Steering Group and seek to expand our industry portfolio via CIKTN and existing contacts. In the long term, as well as the cost and environmental savings our new technologies could bring, there may be a positive impact on public health via cheaper and more available products. We take the opportunity for engagement and leverage at the annual Formulation Engineering Conference (FEC) held at Birmingham, where we will hold a separate spin-out seminar in years 1 and 2 of the project to which all key stakeholders including the above contacts are invited. Through the Natural Products and Drug Discovery undergraduate and MSC module at UEA, which Dr Goss coordinates, there are existing interactions with GSK (Richard Hartley), Astra Zeneca (Duncan Gill and Andy Wells), Novartis (Brian Cox) Novacta (Mike Dawson, Antony Appleyard) and Biotica (Barrie Wilkinson). This network will provide a good initial point for dissemination of research and fine-tuning of the programme as the research progresses in order to meet the needs of industry and SMEs. We will hold a Research Showcase Event in the final year of the project to disseminate results of the project to Industry and promote exploitation (resource is requested to support this event). Training and Investment in People (Jobs) (0-3+ years). This research presents the opportunity for exchanges within the research team to develop novel research ideas and shared knowledge beyond the traditional discipline boundaries. Simmons and Goss have already performed exchanges via discipline hopping project and the development of the EBC concept is directly attributable to this. The continued exchange of personnel between laboratories is critical to the success of the project and builds a highly capable research workforce with excellent employment potential. Further knowledge exchange will be accomplished by -weekly 'sheep-dips' -month long exchanges 'marinades' Additional Communication Resources (0-3 years). We will develop a website to disseminate the work which will be linked from websites accessed by stakeholders including IChemE Biochemical Engineering and Pharma Subject Groups, and Royal Society of Chemistry interest group pages including those of the BioOrganic Group as well as inputting into Knowledge Transfer Networks such as CIKTN.