Skip to main content

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

Periodic Reporting for period 2 - CHASSY (Model-Based Construction And Optimisation Of Versatile Chassis Yeast Strains For Production Of Valuable Lipid And Aromatic Compounds)

Reporting period: 2018-06-01 to 2019-11-30

CHASSY will unlock the full potential of the yeasts Saccharomyces cerevisiae, Yarrowia lipolytica and Kluyveromyces marxianus as cell factories for production of high value compounds for the cosmetic, nutraceutical and white biotechnology sectors. Current cell factory strains for these classes of product are restricted to proof-of-principle levels because of limited precursor supply, poor product tolerance, and lack of versatility. CHASSY addresses these challenges by redesigning metabolic circuits and expanding the host range to include the oleaginous yeast, Y. lipolytica, and the thermotolerant yeast, K. marxianus. The chassis strains will be used to build cell factories to produce three specific high value oleochemicals and aromatics. The major S&T outcomes of this project will be:
(1) a new set of chassis yeast strains with the potential to be developed into industrial cell factories for a range of applications;
(2) the knowledge and technology to readily build and evaluate new chassis tailored to specific applications;
(3) prototype cell factory strains producing three high value metabolites for commercialisation.
CHASSY is also implementing a dissemination and knowledge transfer strategy to ensure that European SMEs benefit from the knowledge base, platform chassis and resources generated in the project.
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 (EC-GEMs) were generated for the three yeast species (S. cerevisiae, K. marxianus and Y. lipolytica) and are being used to design optimal engineering strategies to increase the level of limiting precursors required for production of oleochemicals and aromatics. Semi-absolute proteomics quantification datasets were also generated for the three yeasts under several experimental conditions and an algorithm developed for integration of the proteomics datasets into enzyme-constrained GEMs. In total, 11 condition-specific models with proteomics data were generated for the investigation of metabolic flux distributions of the three yeasts subjected to the various experimental conditions (elevated temperature, low pH and increased osmotic pressure). 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. Standardized toolkits are another output of this ‘build’ objective and are available to the scientific community at Addgene. The toolkits consist of a repository of standardised DNA parts (promoters, genes, terminators, replication origin, selection markers, and integration sites) for assembly of synthetic constructs. This facilitates combinatorial pathway assembly and straightforward incorporation in chassis hosts.

Using the metabolic engineering strategies suggested by the GEMs, and the improved expanded synbio 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. 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 has been used to construct chassis platform strains optimized for the production of fatty acids and long chain lipids.

Applying the yeast chassis platform strains, prototypes that make two oleochemicals (octanoic acid and docosanol) and one aromatic molecule were developed. These strains are now moving into industrial-scale validation and testing. Strains have already been tested under batch bioreactor conditions and are undergoing process development This includes optimisation for industrial medium, scale-up and preliminary downstream processing.

A comprehensive set of transcriptome and proteome data that was generated under industrially relevant stress conditions from cultures grown in chemostat conditions was mined to identify regulatory networks that contribute to stress tolerance. The changed pattern of gene expression after initial response and adaption to the stress was assessed. The most interesting finding was that there is neither a conserved long-term stress response within a species for different stresses, nor between species for the same stress. Instead, evolutionarily-young genes are enriched in the response. These are genes that arose from gene duplications or that recently emerged in a species. There is an important and exciting evolutionary aspect to this as it points to the mechanism by which niche adaption takes please. From a biotechnological viewpoint, it means that the genes that will play a role in long-term robustness are likely to be these evolutionarily-young genes and this is where CHASSY will focus its efforts to further improve chassis strains.
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. Biologically, the most significant output has been the identification of genes involved in niche adaption and this has clear implications for evolutionary biology.

We have developed platform chassis strains for the production of both aromatics and lipids and expect to demonstrate the potential with prototype products in the final research period. 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 hope to overcome some of these limitations to offer opportunities to companies, both large and small, to develop innovative products and processes.
Project Logo
Project page header