Periodic Reporting for period 2 - BIOS (BIOS: The bio-intelligent DBTL cycle, a key enabler catalysing the industrial transformation towards sustainable biomanufacturing)
Reporting period: 2024-04-01 to 2025-09-30
We are all witnessing the consequences of global climate change. To prevent further rise in atmospheric CO2 levels, resources from fossil fuels should remain unused. The implementation of a circular economy is the only way to sustain a high standard of living for the current and the next generation.
To access the products of interest via bioprocesses, typically multiple heterologous genes must be implemented into microbial production hosts. Whereas synthetic biology provides a multitude of novel tools for strain engineering, their rapid and effective implementation in microbial chassis for optimal performance under industrial conditions is still very challenging.
The bio-intelligent approach in BIOS aims to accelerate and improve the conventional ‘design-build-test-learn’ (DBTL) cycle for integrated strain and bioprocess engineering underpinning. Novel innovative metrics, biosensors and bioactuators are developed and fully integrated in the automation platform. Digital twins are created mimicking cellular and process levels. By tightly intertwining AI, not only the prediction quality is improved but, crucially, hybrid learning is made possible. The power of biDBTL will be showcased by creating P. putida producer strains for terpenes, renewable-based polyesters, and methylacrylate.
To access the products of interest via bioprocesses, typically multiple heterologous genes must be implemented into microbial production hosts. Whereas synthetic biology provides a multitude of novel tools for strain engineering, their rapid and effective implementation in microbial chassis for optimal performance under industrial conditions is still very challenging.
The bio-intelligent approach in BIOS aims to accelerate and improve the conventional ‘design-build-test-learn’ (DBTL) cycle for integrated strain and bioprocess engineering underpinning. Novel innovative metrics, biosensors and bioactuators are developed and fully integrated in the automation platform. Digital twins are created mimicking cellular and process levels. By tightly intertwining AI, not only the prediction quality is improved but, crucially, hybrid learning is made possible. The power of biDBTL will be showcased by creating P. putida producer strains for terpenes, renewable-based polyesters, and methylacrylate.
The BIOS project has advanced both the scientific basis and the technical toolbox for bio-intelligent biomanufacturing. On the modelling side, the consortium has built detailed “digital twins” of Pseudomonas putida that combine mechanistic descriptions of metabolism and gene expression with data-driven models. These twins can predict how genetic changes and process conditions influence cell performance and product formation, providing a much stronger basis for rational strain design and model-guided process optimisation.
Experimentally, BIOS has established an integrated platform for building and testing strains. This includes a modern genome engineering toolkit (CRISPR-based control, efficient DNA insertion and recombineering), high-throughput electroporation hardware for rapid transformation in microtiter plates, and optimised cell-free protein synthesis protocols for fast part screening. In parallel, robust fluorescent reporter systems and automated RNA-sequencing workflows have been implemented to quantify gene expression and stress responses in a systematic, reproducible way and to feed these data directly back into the design–build–test–learn cycle.
These capabilities have already led to concrete biological and process advances. BIOS has identified the main stress factors that limit terpene and C1-based production in P. putida and uncovered hidden loss of production capacity over time in lycopene strains, guiding new engineering strategies. For methacrylate esters and other target molecules, new process concepts such as in situ product removal have delivered large increases in titres and much simpler downstream processing. Finally, life-cycle and techno-economic assessments are being used to benchmark these new routes against conventional production, ensuring that the technical innovations are aligned with environmental performance and future industrial viability.
Experimentally, BIOS has established an integrated platform for building and testing strains. This includes a modern genome engineering toolkit (CRISPR-based control, efficient DNA insertion and recombineering), high-throughput electroporation hardware for rapid transformation in microtiter plates, and optimised cell-free protein synthesis protocols for fast part screening. In parallel, robust fluorescent reporter systems and automated RNA-sequencing workflows have been implemented to quantify gene expression and stress responses in a systematic, reproducible way and to feed these data directly back into the design–build–test–learn cycle.
These capabilities have already led to concrete biological and process advances. BIOS has identified the main stress factors that limit terpene and C1-based production in P. putida and uncovered hidden loss of production capacity over time in lycopene strains, guiding new engineering strategies. For methacrylate esters and other target molecules, new process concepts such as in situ product removal have delivered large increases in titres and much simpler downstream processing. Finally, life-cycle and techno-economic assessments are being used to benchmark these new routes against conventional production, ensuring that the technical innovations are aligned with environmental performance and future industrial viability.
The BIOS project has delivered several results that go clearly beyond current practice in biomanufacturing. It has built “digital twins” of Pseudomonas putida that couple mechanistic models of metabolism and gene expression with fast machine-learning surrogates. These models predict the impact of genetic changes and process conditions on growth and product titres far more efficiently than conventional approaches, enabling more targeted strain design and process optimisation.
Experimentally, BIOS has established an integrated genome-engineering and screening platform for P. putida, including CRISPR-based control, efficient DNA insertion methods and automated liquid-handling. A key technical outcome is the iPROBE high-throughput electroporation device for parallel transformation in microtiter plates with induction-based inline sterilisation, for which a patent application has been filed. Together with fluorescent reporters and automated RNA-seq, this allows high-quality data on stress and gene expression to feed directly into the design–build–test–learn cycle.
Scientifically, the project has identified oxidative and membrane stress as major limits for C1 and terpene production and shown that P. putida can consume isoprenol and lycopene, informing pathway design. For methacrylate esters, an in situ product removal concept achieved around 20-fold higher titres and nearly complete product export. Life-cycle and techno-economic analyses highlight solvents and energy use as main impact drivers and guide the Safe- and Sustainable-by-Design development of these new processes.
Experimentally, BIOS has established an integrated genome-engineering and screening platform for P. putida, including CRISPR-based control, efficient DNA insertion methods and automated liquid-handling. A key technical outcome is the iPROBE high-throughput electroporation device for parallel transformation in microtiter plates with induction-based inline sterilisation, for which a patent application has been filed. Together with fluorescent reporters and automated RNA-seq, this allows high-quality data on stress and gene expression to feed directly into the design–build–test–learn cycle.
Scientifically, the project has identified oxidative and membrane stress as major limits for C1 and terpene production and shown that P. putida can consume isoprenol and lycopene, informing pathway design. For methacrylate esters, an in situ product removal concept achieved around 20-fold higher titres and nearly complete product export. Life-cycle and techno-economic analyses highlight solvents and energy use as main impact drivers and guide the Safe- and Sustainable-by-Design development of these new processes.