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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - CHASSY (Model-Based Construction And Optimisation Of Versatile Chassis Yeast Strains For Production Of Valuable Lipid And Aromatic Compounds)

Teaser

Summary

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.

Work performed

One of the key outputs of CHASSY will be yeast strains that produce two oleochemicals (octanoic acid and docosanol) and one plant-derived aromatic to TRL5. These will be valuable products in their own right and will also serve as exemplars and prototypes for these classes of molecules. Improvement of Genome Scale Metabolic Models (GEMs) to provide a greater understanding of cellular metabolism is an important action to achieve this. Subsequently, genes responsible for improved performance under industrial conditions will be identified and synthetic biology tools will be used to build these improved strains. A new GEM concept called GECKO (https://github.com/SysBioChalmers/GECKO) was implemented in CHASSY. Currently available GEMs generally assume that the uptake rate of the carbon source (e.g. glucose) limits production of metabolites of interest in a cell but, in most cases, metabolic fluxes are also limited by their corresponding enzyme levels. GECKO uses enzyme kinetics and protein abundances to constrain a GEM to biologically feasible fluxes. The GECKO method is the first modelling framework to be developed with this capability, vastly improving the predictive power of GEMs. Three new enzyme-constrained genome-scale metabolic models (EC-GEMs) have been generated for the three yeast species (S. cerevisiae, K. marxianus and Y. lipolytica) and are now being used to design optimal engineering strategies to increase the level of limiting precursors required for production of oleochemicals and aromatics.

Systems biology based metabolic engineering relies on DBTL (Design-Build-Test-Learn) and synthetic biology tools are crucial for the â€˜Buildâ€™ component of the cycle. Construction of improved strains necessitates extensive rewiring of central carbon metabolism and this requires highly efficient genome editing methods. CHASSY has already expanded the synbio toolbox for all three yeast strains with significant additional improvements and refinements expected over the lifetime of the project. CRISPRCas9 tools have been developed as an efficient tool for single and multiple-genome editing in K. marxianus and Y. lipolytica. Multiplexing has been achieved in S. cerevisiae with FnCpf1 and is progressing in the other two yeast strains. Finally, a repository of standardised DNA parts (promoters, genes, terminators, replication origin, selection markers, and integration sites) for assembly of synthetic constructs has been created, to facilitate combinatorial assembly and straightforward incorporation in chassis designs.

In addition to improved precursor supply, yeast cell-factory strains need to be robust and stress tolerant. Omics data generated under industrially relevant stress conditions from cultures grown in chemostat conditions is being mined to identify regulatory networks that contribute to stress tolerance. In addition to stress tolerance, yeast strains also need to be tolerant to accumulation of the product of interest. Each of the chassis yeasts was subjected to Adaptive Laboratory Evolution (ALE) and this has resulted in the identification in all strains of genetic mutations that confer growth advantage under particular stress conditions that pertain to industrial production.

Production strains for the three chemicals are being constructed initially in S. cerevisiae to prove the concept and will later be transferred to chassis strains of all three species. Enzyme engineering of Fatty Acid Synthase has resulted in increased production of fatty acids, specifically C8 (octanoic acid) and transporters have been modified to improve export capacity. A sensor for octanoic acid has also been constructed which will be used to screen for high C8-producing strains. For docosanol, strategies have been focused on engineering elongases for preferential production of the very long chain fatty acid (VLCFA) C22 and on identifying reductases that most efficiently convert C22 VLCFA to docosanol. In S. cerevisiae, strains that produce do

Final results

CHASSY will mark the first sustainable production at competitive prices of Octanoic acid and the first viable production Docosanol and of certain aromatics. It will develop a suite of chassis platform yeasts that can be optimised to produce different oleochemicals and aromatic molecules. We will achieve this by using effective, targeted genome engineering and the capacity to recode the genomes of the three yeast species using multiplexing (editing multiple genes simultaneously).

CHASSY will also expand our knowledge of the three target yeast species in a range of ways:
- By advancing the potential of modelling to inform the design of industrial platform yeasts
- By applying ribosome profiling to identify translationally-active mRNAs
- By understanding better the complex catalytic cycle of fatty acid synthases
- By understanding better the regulation of fatty acid and aromatic synthesis at the enzymatic level.
- By clarifying the regulatory networks responsible for stress adaptation at the system level. These mechanisms will be transferrable between yeasts and possibly beyond.

CHASSY also expects to create new market opportunities for environmentally sustainable products and processes; to grow markets by helping to change consumer sentiment; to produce highly trained researchers to fuel the innovation sector; to promote innovation in other sectors (e.g. infection biology; fermented foods and beverages; marine biology). A major goal of the project is to increase awareness of and positive sentiment towards synthetic biology, genetic engineering, and biotechnology across European society.