Biologia Sintética aplicada ao desenvolvimento de fábricas celulares microbianas para produção de anilina

Establishing bioprocesses to produce small compounds, typically obtained through chemical means, has emerged as a promising alternative to reduce reliance on fossil fuels and reduce carbon emissions (Spekreijse, Jurjen; Lammens et al., 2019). Unlike naturally occurring organic alcohols and acids, the bioproduction of aromatic chemicals like aniline presents significant challenges due to their complex molecular structure and the absence of natural synthetic pathways (Winter & Meys, 2021). To date, Covestro stands as the only company to introduce a semi-biological process for aniline production, recently announcing the world's first pilot plant for biobased aniline (Jaeger et al., 2017). This process involves partial bioprocessing, with the engineered microorganism converting renewable substrates into an aniline precursor, subsequently chemically catalyzed into aniline. The LCA of bio-based aniline production indicates a substantial reduction in global warming impacts, ranging from 35% to 69%, depending on the raw material used (Winter & Meys, 2021), underscoring the potential of biobased aniline as a sustainable alternative. Bondalti, the leading Portuguese chemical company, recognizes the market potential, emphasizing the urgency of developing sustainable processes. The shift towards more sustainable practices in a circular economy framework underscores the need for microbial cell factories capable of efficiently converting renewable raw materials into bulk chemicals. However, achieving an efficient bioprocess necessitates a holistic approach encompassing pathway optimization, organism engineering, and detailed process conditions. To address this challenge comprehensively, we aim to develop a microbial cell factory using Escherichia coli to convert renewable feedstocks into aniline. Our multidisciplinary team, comprising members from the Systems and Synthetic Biology and Protein Modeling Labs at ITQB-NOVA, supported by an academic consultant and industrial experts from Bondalti, is poised to pioneer a fully biological aniline production process that meets economic and environmental criteria. To achieve this ambitious objective, different goals must be addressed: - Generate biosynthetic pathways to produce aniline by enumeration and retrosynthesis algorithms expanding nature’s portfolio. - Select the most promising pathway favoring minimal size and maximum carbon conservation. - Integrate the most promising vias into metabolic and dynamic models of E. coli to find an optimum genotype. - Simulate the growth of the engineered strains in different media mimicking the formulation of various biomass hydrolysates to identify optimal growth conditions leading to maximum product yield. - Select the most suitable gene candidates for each reaction using the Gene Discovery and Enzyme Engineering pipeline. - For complex reactions, apply Enzyme Engineering methods to find mutants with increased activity towards the target substrate. - Implement in vivo the selected pathways and the optimum genotype. - Evolve the strains to increase the consumption yield of the selected renewable substrates. - Characterize phenotypically the developed cells in bioreactors. - Simulate and design the plant of the bioprocess with process simulation software and feed-back and -forward with the strain design tasks to establish realistic targets for the novel strains. - Evaluate the environmental impact of the bioprocess using the principles of the LCA methodology.