All of the publications that have resulted from the CHASSY project are also available on the project ZENODO page, which you can access here.
Analysis of the first round of omics data from the CHASSY project has revealed that robustness is likely to be multifactorial. By leveraging gene age information, it will be possible to focus rational experimental designs on unpredicted stress tolerance genes, which prior to this work fall into the category of genes of unknown function. Further characterization of young genes from species that thrive in harsh environments can inform the design of more robust strains for biotechnology. This was a highly collaborative piece of work that demonstrates the significant benefits of a multi-parnter consortium. You can access the full article in Nature Communications here.
Yarrowia lipolytica is becoming more widely used as a microbial cell factory chassis in the development of industrial applications aiming to produce fatty acids, organic acids, or enzymes. This is a direct result of the improvements that are being made in tools for genome editing in this yeast. This paper from INRAE Paris describes the construction and validation of a set of Yarrowia lipolytica CRISPR/Cas9 vectors containing six different markers that allows virtually any genetic background to be edited. The Golden Gate method was used to assemble the vectors, which are now available via Addgene (129,656–129,661). In all these vectors, any gRNA sequence can be easily and rapidly introduced using Golden Gate assembly. You can access the full article in Biotechnology Letters here.
De novo biosynthesis of 8-hydroxyoctanoic acid via a medium-chain length specific fatty acid synthase and cytochrome P450 in Saccharomyces cerevisiae
Scientists at Goethe University Frankfurt have previously described short chain fatty acid production in bakers yeast. They have taken this work a step further to produce terminally hydroxylated fatty acids or dicarboxylic acids, industrially relevant compounds with broad applications. In this article Wernig at al., describe proof of principle for the de novo biosynthesis of 8-hydroxyoctanoic acid from glucose and ethanol in the yeast Saccharomyces cerevisiae. The authors have constructed a complete pathway from glucose or ethanol towards 8-hydroxyoctanoic acid by combining octanoic acid production via a mutated FAS with direct ω-hydroxylation of the octanoic acid by various cytochrome P450s. You can access the full article in Metabolic Engineering Communications here.
Origin of Lactose Fermentation in Kluyveromyces lactis by Interspecies Transfer of a Neo-functionalized Gene Cluster during Domestication
The Yeast Biotechnology Research Group at UCC are experts on the industrial yeast Kluyveromyces marxianus. In collaboration with scientists from TU Delft and UCD they provide a fascinating insight into the selective pressures imposed on yeasts by early farmers for the ability to ferment animal milk. This paper describes how an insect-associated, lactose negative progenitor of the milk yeast K. lactis acquired the genes that enable lactose fermentation from a dairy-adapted population of K. marxianus. You can access the full text paper in Current Biology here
A consensus S. cerevisiae metabolic model Yeast8 and its ecosystem for comprehensively probing cellular metabolism
The Nielsen group at Chalmers University of Technology are leaders in the field of metabolic modelling. In this paper the team presents Yeast8, the latest release of the consensus GEM of S. cerevisiae. They also introduce ecYeast8, a model incorporating enzyme constraints; panYeast8 and coreYeast8, representing the pan and core metabolic networks of 1011 S. cerevisiae strains; and proYeast8DB, a database containing 3D structures of metabolic proteins. You can access the full text paper in Nature Communications here.
Connecting central carbon and aromatic amino acid metabolisms to improve de novo 2-phenylethanol production in Saccharomyces cerevisiae
Jasmijn and the team at TU Delft have done considerable work on improving de novo 2-phenylethanol production. They have generated yields of that are higher than anything previously reported in S. cerevisiae and other yeast species. By improving phosphoenolpyruvate supply and eliminating the formation of p-hydroxy-phenylethanol significant improvements to 2PE production in S. cerevisiae were achieved. These mutations combined with the engineering of the aromatic amino acid biosynthesis and Ehrlich pathway, optimize carbon flux towards 2PE. You can access the full text paper in Metabolic Engineering here.
Macarena Larroudé and her colleagues at INRA have developed a new modular Golden Gate toolkit for the one-step assembly of three transcription units that includes a selective marker and sequences for genome integration in Y. lipolytica. This powerful new tool will enable biologists to properly engineer this industrial yeast and take full advantage of its potential in bio-based production of valuable compounds. This efficient, standardized toolbox is available to the community through AddGene. You can access the full text paper in Microbial Biotechnology here.
The CHASSY teams at University College Cork and Technical University Delft are making it easier to engineer K. marxianus as a next generation cell factory for bio-based chemicals. This non-conventional yeast has many physiological and metabolic traits that make it a very promising cell factory for a range of bio-based products. However, even though the wild type strain is already used to produce fragrances and fermented products, a lack of standardised tools has hindered its use in many other spheres. This paper brings together a set of parts for the expression of multiple genes for metabolic engineering and synthetic biology. You can find the relevant plasmids on AddGene.
The CHASSY team at University College Cork examined the Major Superfamily Transporters for sugar in the industrial yeast, Kluyveromyces marxianus. The focus was on the sugar galactose since it was already reported in K. lactis that this hexose was a substrate for both Lac12 and Hgt1. It emerged that three of the four copies of Lac12, four Hgt-like proteins and one Kht-like protein have some capacity to transport galactose when expressed in S. cerevisiae and inactivation of all eight genes was required to completely abolish galactose uptake in K. marxianus. The data highlight how gene duplication and functional diversification has provided K. marxianus with versatile capacity to utilise sugars for growth. Read more here.
Short and medium chain fatty acids (SMCFA) are important as platform chemicals, however, they are usually produced from unsustainable resources. They can be produced in engineered microbial cells, but a biosensor to screen for the best-producing cells is required. In this paper, CHASSY partners in GUF and UCC present their whole-cell biosensor for rapid detection of SMCFA. Validated using octanoic acid producing strains, this biosensor will enable high-throughput screening of SMCFA producers and could drastically speed up the engineering of SMCFA producting cell factories. Read the full paper in ACS Synthetic Biology here.
Metabolic engineering of Saccharomyces cerevisiae for production of very long chain fatty acid-derived chemicals
Microbial fermentation using engineered yeast to produce chemicals and biofuels is an economical and sustainable alternative to traditional chemical synthesis from petroleum. Here, partners from the CHASSY project in Chalmers and Biopetrolia and their colleagues present the construction of a S. cerevisiae platform strain for high-level production of very-long-chain fatty acid (VCLFA)-derived chemicals. Their approach will provide a universal strategy towards the production fo similar high value chemicals in a scalable, stable and sustainable manner. Read the full paper in Nature Communications here.
Yarrowia lipolytica is one of the three target yeast species in the CHASSY project. It has recieved an increasing amount of research attention in recent years as a promising cell factory for the production of compounds of industrial interest. It is a good natural producer of citric acid, erythritol, various proteins and lipids. Y. lipolytica can grow at high densities and produce large titers of product. Recent advances in synthetic biology have rapidly increased the ability of researchers to engineer this yeast to produce an expanded range of valuable compounds and to increase its robustness to withstand harsh industrial conditions and processes. Read a review of the latest SynBio tools developed for Y. lipolytica published in Biotechnology Advances here.
Fatty alcohols can be used for, among other applications, biofuels. Yeast has high potential for industrial scale production of these valuable compounds, but accumulation of fatty alcohols can impair yeast growth rates and extraction can be costly. In this paper, colleagues from Chalmers University of Technology demonstrate that certain heterologous transporters may facilitate successful commercialization of fatty alcohol production in yeast. This could inspire the design of novel cell factories. Read the abstract here: Show abstract...
Genome editing in Kluyveromyces and Ogataea yeasts using a broad-host-range Cas9/gRNA co-expression plasmid
This paper is a collaboration between colleagues in Delft and UCC. They have constructed a plasmid that has shown to be efficient in deactivating the ADE2 gene in four different yeast species. This could open up new paths of research in non-conventional yeasts as previous plasmids and cassettes fro Cas9 and guide-RNA expression were species-specific. The research was published in FEMS Yeast Research.
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Under pressure: evolutionary engineering of yeast strains for improved performance in fuels and chemicals production
Evolutionary engineering, which uses laboratory evolution to select for industrially relevant traits, is a popular strategy in the development of high-performing yeast strains for industrial production of fuels and chemicals. By integrating whole-genome sequencing, bioinformatics, classical genetics and genome-editing techniques, evolutionary engineering has also become a powerful approach for identification and reverse engineering of molecular mechanisms that underlie industrially relevant traits. New techniques enable acceleration of in vivo mutation rates, both across yeast genomes and at specific loci. Recent studies indicate that phenotypic trade-offs, which are often observed after evolution under constant conditions, can be mitigated by using dynamic cultivation regimes. Advances in research on synthetic regulatory circuits offer exciting possibilities to extend the applicability of evolutionary engineering to products of yeasts whose synthesis requires a net input of cellular energy. The review is available to read in Current Opinion in Biotechnology.
CHASSY PI, Jean-Marc Daran of TU Delft and his colleagues have experimented with using a new type of CRISPR technology for genome editing in the industrial yeast, Saccharomyces cerevisiae. They found Cpf1 to be highly efficient at introducing point mutations with high fidelity, and multi-gene editing. The system was also efficient at promoting recombination of the repair fragment.
Improving the phenotype predictions of a yeast genome-scale metabolic model by incorporating enzymatic constraints
Genome-scale metabolic models (GEMS) are a useful tool for calculating metabolic phenotypes. Research by CHASSY PI Jens Nielsen and his colleagues have improved the GEMs for Saccharomyces cerevisiae by applying GECKO, a method that also accounts for enzymes as part of reactions. This method is expected to increase the use of model-based design in metabolic engineering. The research is available to read in Molecular Systems Biology.
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