17-ERACoBioTech: Fabrication of hierarchically organized multi-functional heterogeneous biocatalysts

Chemical synthesis catalyzed by enzymes is contributing to establish a modern chemistry supported on cleaner, faster and safer chemical reactions. In particular, cell-free synthetic biology (or systems biocatalysis) in solution is currently emerging as an attractive alternative to synthetic biology using whole cells because isolated enzymes do not present regulation constraints at genomic level and the intensification of the chemical fluxes do negligible effect on the system subsistence. However, it presents major issues in terms of both process- and cost-efficiency because these soluble systems often reach low chemical yields, are notably unstable and their re-usability is rather limited. In order to overcome these limitations, this proposal aims to assemble multi-enzyme systems at the nanoscale of solid and porous materials aided by protein scaffolds that guarantee the hierarchical and spatial organization of the functional modules. This immobilized multi-enzyme cascade will be utilized as heterogeneous multi-functional biocatalyst to transform renewable raw materials into omega-aminoacids, in one-pot with in situ cofactor regeneration. The fabrication and exploitation of such heterogeneous biocatalyst will be achieved by i) engineering the proposed artificial pathway for the synthesis of omega-aminoacids using bio-oils and diols as raw materials, ii) engineering a modular protein (consensus tetratricopeptide repeat: CTPR) as scaffolding unit to organize the multi-enzyme system at the nanoscale, iii) in vitro assembling of multi-enzyme systems onto the CTPR scaffolds, iv) immobilizing such multi-enzyme assemblies on solid particles, v) using the hierarchically organized multi-function heterogeneous biocatalysts for the efficient and sustainable production of long and short omega-aminoacids vi) 10 L scale-up the process under industrially relevant conditions and vii) manufacturing of one modular kit based on DNA plasmids that express assemblies of different multi-enzyme systems and one modular kit that allows the solid-phase assembly of different functional modules based on scaffolded multi-enzyme systems. These new platforms will open an innovative tool to build sustainable pathways for chemical manufacturing of high added value molecules using renewable raw materials. The rational integration of different enzymes as functional modules with an engineered protein scaffold and a porous material as heterogeneous chassis will be addressed by combining protein engineering, surface chemistry and protein immobilization tools. The research team presents a solid and multidisciplinary background in those areas, which fully qualifies this team to carry out this project at the frontier between the chemistry and the biology. Moreover, the research consortium is hosted in different research institutions that provide a suitable research environment (three academic partners; CIC biomaGUNE, University of Nottingham and Ruhr Universität-Bochum, and one industrial partner: Bioassays) to successfully address the major challenges and meet the main objectives of this project.

The main goal of the project is to develop a tool-box for the fabrication of hierarchically organized heterogeneous multi-functional biocatalysts.
As proof of the concept, we have designed two artificial cascades for the synthesis of omega-aminoacids (omega-AA) using diols and bio-oils as renewable raw materials; both cascades will be based on 4 different functional modules (A-D). We will assemble the different enzymes forming modules A-D into protein scaffolds based on the consensus tetratricopeptide repeats (CTPR) to endow such functional modules with nanometric proximity. We will construct a set of plasmid encoding chimeric proteins with functional (enzyme) and structural (scaffold brick) domains in order to assemble 4 functional modules that can be readily immobilized on solid carriers to create a battery of modular heterogeneous biocatalysts. The immobilized functional modules can be combined ad hoc to catalyze one or the other synthetic scheme depending on the available raw materials. We will demonstrate the applicability of the immobilized functional modules by demonstrating and scaling-up the synthesis of long and short -AA. Finally the fabrication of one set of plasmids and one kit for solid-phase and tailor-made assembly of different enzyme-scaffold units will be demonstrated and validated at industrial level.

The proposed work in HOMBIOCAT will have a remarkable positive impact on the aim of transforming the global economy from a dependence on fossil raw materials to a sustainable bio-based economy. In this sense, the proposed technologies based on synthetic biology and biotechnological approaches focused on the development of industrial biotechnologies. In particular, the project responds to three main needs in coincidence with the topics of ERA CoBioTech call: 1. the sustainable production and conversion of bioresources into value-added products ; 2. the production of bio-based value-added products;3. the development of sustainable industrial processes including the scale-up of biotechnological processes.

Who will benefit from this research?
The project aims at developing(TRL 3-4), demonstrating (TRL5) and validating (TRL6) an innovative technology based on modular systems to fabricate multi-functional heterogeneous biocatalysts with potential application in Biotechnology. Therefore Industry will be a key beneficiary but academic will also strongly benefit from the developed technologies.

How will they benefit from this research?
Once the tools for this technology are developed, we will demostrante the operational functionality of these heterogeenous biocatalysts at lab scale, performing the industrially relevant biotransformation for which they have been designed (TRL4). We will scale-up the continuous biosynthesis of omega-aminoacids to industrially relevant substrate concentrations (>100 g/L) and total turnovers numbers of the cofactors (>1000) and enzymes (TTN>10000). Finally, we propose a parallel pathway in the technological exploitation leaded by the industrial partner. BIOA will validate (TRL5) and demonstrate (TRL6) that this generic technology serves as a tool-kit for the integration of any multi-enzyme system into hierarchically organized heterogeneous biocatalysts.


What will be done to ensure that they have the opportunity to benefit from this research?
As well as Open Access publications, presentations at conferences and direct communication with the industrial partners through the consortium, we will ensure that outcomes from this project will be highlighted on our web pages (research group as well as the project website), the University of Nottingham's Communications and Marketing Unit, Nottingham's Café Scientifique and BioCity, and the BBSRC media office. Throughout the project we will also expand our interaction with industrial partners such as Johnson Matthey and Pfizer, and exploit BBSRC NIBBs such as BioCatNet and CBMNet.


Professional development for staff working on the project
The project offers significant opportunities for the postdoctoral researchers involved in the consortium to broaden his/her skills. The overall project is highly interdisciplinary, encompassing chemistry, enzymology and microbiology, and engineering. The PDRA will be exposed to a variety of techniques within the laboratories of both PI and CoI as well as through the network of collaborators. Specifically at Nottingham, we will encourage the development of the PDRA scientific communication skills by regularly presenting our research to peer academic audiences as well as the general public (e.g. Nottingham's Café Scientifique or local schools). The University of Nottingham has a well-established Science Outreach Programme, which will be key in the training of the PDRA in addressing a lay audience, and Dr. Paradisi has also extensive experience in outreach and bringing science to schools. Careers advice for the PDRA will be provided by the University of Nottingham, which runs training courses on career planning, effective CV writing workshops and interview practice for early career scientists.