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

酶催化的化学合成有助于建立以更清洁、更快和更安全的化学反应为基础的现代化学。特别是，溶液中的无细胞合成生物学（或系统生物催化）目前正成为使用全细胞合成生物学的一种有吸引力的替代方法，因为分离的酶在基因组水平上不存在调节限制，并且化学通量的增强对系统生存的影响可以忽略不计。然而，它在工艺和成本效率方面提出了主要问题，因为这些可溶系统通常达到低化学产量，明显不稳定，其可重复使用性相当有限。为了克服这些限制，本提案旨在在蛋白质支架的辅助下，在纳米尺度上组装固体和多孔材料的多酶系统，以保证功能模块的层次和空间组织。这种固定化的多酶级联将被用作多相多功能生物催化剂，将可再生原料转化为omega-氨基酸，在一个锅中进行原位辅因子再生。这种异质生物催化剂的制造和开发将通过以下方式实现：1)设计以生物油和二醇为原料合成ω -氨基酸的人工途径；2)设计模块化蛋白质(共识四肽重复：CTPR)作为支架单元在纳米尺度上组织多酶系统，iii)在体外将多酶系统组装到CTPR支架上，iv)将这些多酶组件固定在固体颗粒上，v)使用分层组织的多功能异质生物催化剂高效和可持续地生产长和短omega-氨基酸vi)在工业相关条件下扩大10 L的工艺；vii)制造一种基于表达不同多酶系统组装的DNA质粒的模块化试剂盒和一种基于支架多酶系统的允许不同功能模块固相组装的模块化试剂盒。这些新平台将为使用可再生原材料的高附加值分子的化学制造开辟一条可持续发展的途径。结合蛋白质工程、表面化学和蛋白质固定工具，将不同的酶作为功能模块，以工程蛋白支架和多孔材料作为异质底盘进行合理整合。该研究团队在这些领域具有坚实的多学科背景，完全有资格在化学和生物学的前沿开展该项目。此外，研究联盟由提供合适研究环境的不同研究机构主持（三个学术合作伙伴：CIC biomaGUNE，诺丁汉大学和鲁尔Universität-Bochum，以及一个工业合作伙伴：Bioassays），以成功解决主要挑战并实现本项目的主要目标。

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.