Periodic Reporting for period 1 - HYDROCOW (Hydrogen oxidizing bacteria engineered to valorize CO2 for whey protein production)
Periodo di rendicontazione: 2023-09-01 al 2024-08-31
Milk protein plays an important role in our nutrition, however classical milk production has significant environmental impacts, from greenhouse gas (GHG) emissions to extensive land demand. Project HYDROCOW addresses these challenges through a net-zero carbon dairy protein production platform. The main objective of the project is to develop and demonstrate a bacterial protein secretion system where CO2, and soon N2, are valorized into food-grade protein, decoupled from agriculture. This system will be based on the first-of-a-kind engineered hydrogen oxidizing bacterium (eHOB) Xanthobacter sp. SoF1. As a first product the main milk component beta-lactoglobulin was chosen. This will be achieved by implementing a Design-Build-Test-Learn (DBTL) cycle linked to a validation and scaleup phase allowing to iteratively optimize the production of secreted protein. The project will deliver key technologies – A) an innovative eHOB protein secretion system; B) predictive eHOB metabolic models, genetic engineering tools, and a novel high-throughput (HTP) screening system for DBTL cycling; and C) the methods for validation and scale-up – with immediate and long-term impact on the production of food and nutrition, materials, medicines, fuels and chemicals. In the long-term, the proposed platform has the potential to not only replace conventionally produced food proteins but also deliver proteins for materials or therapeutics, important for human and animal health. In comparison to current standard microbial production processes our platform does not compete with human nutrition for valuable feedstock, such as glucose, and therefore will contribute to a sustainable development of our society. HYDROCOW will generate significant knowledge for a growing research and application community about autotrophic, microbial production systems, their physiology, and sophisticated tools for genetically designing and screening them.
The goal of project HYDROCOW is to engineer the hydrogen-oxidizing bacterium Xanthobacter sp. SoF1 into a cell factory capable of secreting high levels of beta-lactoglobulin, a primary milk component. We aim to accomplish this through a Design-Build-Test-Learn (DBTL) cycle. In short: using computational modeling, we design engineering strategies, construct these designs in DNA, and test their performance. By iteratively refining our designs, we will optimize the system until it is suitable for validation and production.
In the first year, we constructed a draft genome-scale model of Xanthobacter sp. SoF1. As a prerequisite, we conducted comprehensive genome annotation using a multi-faceted bioinformatics approach. This genome-scale model will support the design of engineering strategies in silico. To validate the genome-scale model, we conducted an extensive survey of metabolizable carbon sources and collected transcriptomic and proteomic datasets from the organism under laboratory and process conditions, as well as during shifts between these states. These datasets are crucial for linking laboratory optimization to later stages of validation and production.
To implement the engineering strategies, we developed a modular cloning toolkit that allows for fine-tuning gene expression levels as needed. We also established workflows for introducing foreign DNA into the strain and for characterizing the toolkit’s performance.
For functional protein secretion testing, we developed and compared various protein detection methods, both labeled and label-free, and integrated them into our high-throughput nanoliter technology. This technology will facilitate the identification of optimal designs, and we started a first screen for finding strong protein expressing Xanthobacter sp. SoF1 variants.
We effectively connected project HYDROCOW to other projects in the Nitrogen and CO2 utilization portfolio by organizing meetings for senior and junior scientists. Further, we already trained two master students and one bachelor student within this multidisciplinary project.
In the first year, we constructed a draft genome-scale model of Xanthobacter sp. SoF1. As a prerequisite, we conducted comprehensive genome annotation using a multi-faceted bioinformatics approach. This genome-scale model will support the design of engineering strategies in silico. To validate the genome-scale model, we conducted an extensive survey of metabolizable carbon sources and collected transcriptomic and proteomic datasets from the organism under laboratory and process conditions, as well as during shifts between these states. These datasets are crucial for linking laboratory optimization to later stages of validation and production.
To implement the engineering strategies, we developed a modular cloning toolkit that allows for fine-tuning gene expression levels as needed. We also established workflows for introducing foreign DNA into the strain and for characterizing the toolkit’s performance.
For functional protein secretion testing, we developed and compared various protein detection methods, both labeled and label-free, and integrated them into our high-throughput nanoliter technology. This technology will facilitate the identification of optimal designs, and we started a first screen for finding strong protein expressing Xanthobacter sp. SoF1 variants.
We effectively connected project HYDROCOW to other projects in the Nitrogen and CO2 utilization portfolio by organizing meetings for senior and junior scientists. Further, we already trained two master students and one bachelor student within this multidisciplinary project.
The genome-scale model that was developed in the first year includes a transcript/protein allocation term and is explicitly suitable for predicting optimal engineering strategies for protein expression and secretion. It allows for direct integration of the various OMICS data that were created under various laboratory and process conditions. Such models will find applications well beyond the scope of HYDROCOW, thus serving as tools for developing any microbial-based bioproduction technology. In combination with the demonstrated (pan)genome analysis pipeline we envision maximizing the utility of limited data for engineering purposes.
Further, for implementing the engineering designs, we identified several promoters that lead to strong protein expression in X. sp. SoF1. This is an important starting point for BGL expression and secretion in the developed screening platform. Additional strong promoters can be extracted from the omics data.
Further, for implementing the engineering designs, we identified several promoters that lead to strong protein expression in X. sp. SoF1. This is an important starting point for BGL expression and secretion in the developed screening platform. Additional strong promoters can be extracted from the omics data.