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Model-Based Construction And Optimisation Of Versatile Chassis Yeast Strains For Production Of Valuable Lipid And Aromatic Compounds

Periodic Reporting for period 3 - CHASSY (Model-Based Construction And Optimisation Of Versatile Chassis Yeast Strains For Production Of Valuable Lipid And Aromatic Compounds)

Reporting period: 2019-12-01 to 2021-05-31

Delivery of a sustainable circular bioeconomy and achievement of key UN SDGs needs efficient microbial cell factories that can replace fossil-resources for the production of high value compounds for the cosmetic, nutraceutical and white biotechnology sectors. CHASSY was established to address a key bottleneck that currently restricts the rate at which new yeast cell factories are commercialised. The problem has been that each new strain needs to be designed and built from scratch – which is slow and inefficient. The first objective of CHASSY was to overcome this by designing chassis strains, or platforms, into which new production pathways could be added. This was achieved in three different industrial yeasts, one suitable for a wide range of applications, a second more adapted to oleochemicals, and a third that is especially suitable for aromatic molecules. The second objective was to establish knowledge and technology to readily build and evaluate new chassis strains. This was accomplished though the development of new computational modelling tools (software) as well as a wide range of new synthetic biology tools and resources. The project also had a goal of building prototype cell factory strains that produce specific high value oleochemicals and aromatics with potential for commercialisation. This was successful and prototypes for both classes of molecules were constructed and evaluated under industrial conditions. The final objective was to offer European SMEs the opportunity to benefit from the knowledge coming from CHASSY. We did this by making many of our resources available on open access formats and by implementing a comprehensive communication and dissemination plan using the full range of media and routes available.
In order to build platform chassis strains, a greater understanding of cellular metabolism is required alongside improved engineering tools. Three new enzyme-constrained genome-scale metabolic models (ecModels) were generated for the three yeast species (S. cerevisiae, K. marxianus and Y. lipolytica). They were used to predict gene targets for metabolic rewiring, aiming to enhance production of octanoic acid, docosanol and naringenin. Metabolic rewiring strategies to promote enhanced accumulation of metabolic precursors such as L-phenylalanine, 2-phenylethanol and acetyl-CoA was also studied using the ecModels. These models have been made available through the open access repository GitHub (https://github.com/SysBioChalmers/ecModels) and are already being used by a number of external research groups. In addition, a user-friendly interface for prediction of engineering targets has been developed which opens up new exploitation routes for industry. This is accessible via the Caffeine project (https://caffeine.dd-decaf.eu/).
To enable the ‘build’ part of the ‘design-build-test-learn’ cycle, genome engineering tools were developed for all three yeasts. Editing tools with multiple CRISPR Cas endonucleases and multiplexing genome editing were implemented. These toolkits are available to the scientific community via Addgene and multiple groups and teams are now using them. Using the metabolic engineering strategies suggested by the ecModels, and the improved expanded synthetic biology toolbox, we were able to optimize the supply of precursors to, and flux through, the shikimate pathway. Therefore, a major outcome of the project is a collection of chassis platform strains that synthesize higher levels of aromatic amino acids. These strains are of interest to the flavour, nutraceutical and personal care sectors. It will now be possible to add further pathways to produce molecules that have commercial potential such as Flavonoids, Stilbenes, Coumarins and Lignans. A similar engineering approach was used to construct chassis platform strains optimized for the production of fatty acids and long chain lipids. By applying the yeast chassis platform strains, several prototypes were developed and tested under industrial-scale conditions. While the results are promising, more optimization is required, e.g. industrial medium, scale-up and preliminary downstream processing, before the process can be moved to full-scale production. Nevertheless, some external collaborations have already been initiated with industry partners to further develop and commercially exploit some of these results with platform strains in both the aromatic and oleochemical space.
Scientific publications formed the backbone of the dissemination strategy for many of these results. CHASSY published a total of 33 peer reviewed scientific articles over the course of the project, including publications in high impact journals such as Nature Energy, Nature Catalysis, Nature Communications, Current Biology and Nucleic Acids Research. These results have also been disseminated at over 60 events over the course of the project. There were also many industry-focused events and extensive dissemination to the SME sector.
CHASSY has already achieved substantial advances beyond the state of the art. The new GEMs are a major progression beyond what was available at the start of the project. A whole suite of new tools, analysis pipelines and data visualization resources were created and made available to the research community via publications, Github and Addgene. The capacity to engineer metabolic pathways in K. marxianus and Y. lipolytica is much greater now as a result of our work and outputs. Although we are using these resources, the larger impact will come from the adoption of our materials by research groups in the academic and industrial research communities.
We developed platform chassis strains for the production of both aromatics and lipids and are on the cusp of demonstrating the potential with prototype products. The project will make an important contribution to the development of the bioeconomy. Industrial biotechnology can help deliver a sustainable solution to the challenge of replacing fossil resources with bio-based ones. The sector is hampered, however, by the lack of success stories and high technological entry barriers, and the related associated cost. With CHASSY, we are overcoming these limitations to offer opportunities to companies, both large and small, to develop innovative products and processes. The project has been a tremendous success.
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