All of the publications that have resulted from the CHASSY project are also available on the project ZENODO page, which you can access here.

Synthetic biology tools for engineering Y. lipolytica

Synbio Y lipolytica snipYarrowia 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 written by CHASSY researchers at INRA Ile-de-France and their colleagues here.



transporter expression snip Heterologous transporter expression for improved fatty alcohol secretion in yeast

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... ABSTRACT: The yeast Saccharomyces cerevisiae is an attractive host for industrial scale production of biofuels including fatty alcohols due to its robustness and tolerance towards harsh fermentation conditions. Many metabolic engineering strategies have been applied to generate high fatty alcohol production strains. However, impaired growth caused by fatty alcohol accumulation and high cost of extraction are factors limiting large-scale production. Here, we demonstrate that the use of heterologous transporters is a promising strategy to increase fatty alcohol production. Among several plant and mammalian transporters tested, human FATP1 was shown to mediate fatty alcohol export in a high fatty alcohol production yeast strain. An approximately five-fold increase of fatty alcohol secretion was achieved. The results indicate that the overall cell fitness benefited from fatty alcohol secretion and that the acyl-CoA synthase activity of FATP1 contributed to increased cell growth as well. This is the first study that enabled an increased cell fitness for fatty alcohol production by heterologous transporter expression in yeast, and this investigation indicates a new potential function of FATP1, which has been known as a free fatty acid importer to date. We furthermore successfully identified the functional domain of FATP1 involved in fatty alcohol export through domain exchange between FATP1 and another transporter, FATP4. This study may facilitate a successful commercialization of fatty alcohol production in yeast and inspire the design of novel cell factories.



genome editing kluyveromycesGenome 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.

Read the abstract here: Show abstract... ABSTRACT: While CRISPR-Cas9-mediated genome editing has transformed yeast research, current plasmids and cassettes for Cas9 and guide-RNA expression are species specific. CRISPR tools that function in multiple yeast species could contribute to the intensifying research on non-conventional yeasts. A plasmid carrying a pangenomic origin of replication and two constitutive expression cassettes for Cas9 and ribozyme-flanked gRNAs was constructed. Its functionality was tested by analyzing inactivation of the ADE2 gene in four yeast species. In two Kluyveromyces species, near-perfect targeting (≥96%) and homologous repair (HR) were observed in at least 24% of transformants. In two Ogataea species, Ade−mutants were not observed directly after transformation, but prolonged incubation of transformed cells resulted in targeting efficiencies of 9% to 63% mediated by non-homologous end joining (NHEJ). In an Ogataea parapolymorpha ku80 mutant, deletion of OpADE2 mediated by HR was achieved, albeit at low efficiencies (<1%). Furthermore the expression of a dual polycistronic gRNA array enabled simultaneous interruption of OpADE2 and OpYNR1 demonstrating flexibility of ribozyme-flanked gRNA design for multiplexing. While prevalence of NHEJ prevented HR-mediated editing in Ogataea, such targeted editing was possible inKluyveromyces. This broad-host-range CRISPR/gRNA system may contribute to exploration of Cas9-mediated genome editing in other Saccharomycotina yeasts.



Under pressure snipUnder 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.

FnCpf1snipFnCpf1: a novel and efficient genome editing tool for Saccharomyces cerevisiae

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.


The published research is available to read in Nucleic Acids Research. You can read the abstract here: Show abstract... ABSTRACT: Cpf1 is a new class II family of CRISPR-Cas RNA-programmable endonucleases with unique features that make it a very attractive alternative or complement to Cas9 for genome engineering. Using constitutively expressed Cpf1 from Francisella novicida, the present study demonstrates that FnCpf1 can mediate RNA-guided DNA cleavage at targeted genomic loci in the popular model and industrial yeast Saccharomyces cerevisiae. FnCpf1 very efficiently and precisely promoted repair DNA recombination with efficiencies up to 100%. Furthermore, FnCpf1 was shown to introduce point mutations with high fidelity. While editing multiple loci with Cas9 is hampered by the need for multiple or complex expression constructs, processing itself a customized CRISPR array FnCpf1 was able to edit four genes simultaneously in yeast with a 100% efficiency. A remarkable observation was the unexpected, strong preference of FnCpf1 to cleave DNA at target sites harbouring 5′-TTTV-3′ PAM sequences, a motif reported to be favoured by Cpf1 homologs of Acidaminococcus and Lachnospiraceae. The present study supplies several experimentally tested guidelines for crRNA design, as well as plasmids for FnCpf1 expression and easy construction of crRNA expression cassettes in S. cerevisiae. FnCpf1 proves to be a powerful addition to S. cerevisiae CRISPR toolbox.


GEM snipImproving 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.

You can read the abstract here: Show abstract... ABSTRACT: Genome-scale metabolic models (GEMs) are widely used to calculate metabolic phenotypes. They rely on defining a set of constraints, the most common of which is that the production of metabolites and/or growth are limited by the carbon source uptake rate. However, enzyme abundances and kinetics, which act as limitations on metabolic fluxes, are not taken into account. Here, we present GECKO, a method that enhances a GEM to account for enzymes as part of reactions, thereby ensuring that each metabolic flux does not exceed its maximum capacity, equal to the product of the enzyme’s abundance and turnover number. We applied GECKO to a Saccharomyces cerevisiae GEM and demonstrated that the new model could correctly describe phenotypes that the previous model could not, particularly under high enzymatic pressure conditions, such as yeast growing on different carbon sources in excess, coping with stress, or overexpressing a specific pathway. GECKO also allows to directly integrate quantitative proteomics data; by doing so, we significantly reduced flux variability of the model, in over 60% of metabolic reactions. Additionally, the model gives insight into the distribution of enzyme usage between and within metabolic pathways. The developed method and model are expected to increase the use of model-based design in metabolic engineering.

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