Yeast-based screening platforms to understand and improve human health

Deichmann, Marcus; Hansson, Frederik G.; Jensen, Emil D.

doi:10.1016/j.tibtech.2024.04.003

Cited by 5 publications

(3 citation statements)

References 138 publications

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“…As shown here, the HEEL methodology can minimize the problem of phenotypic noise, i.e., having multiple genotypes present in each cell during a mutant yeast screening. Our automated genotyping workflow allows for hundreds of yeast cells to be efficiently genotyped, which significantly simplifies the identification of beneficial mutants when using yeast as a screening platform (9). In addition, while the number of mutants screened and genotyped would be limited to just a few hundred, predictive algorithms should nevertheless be able to generate more insightful models from this smaller, but more high-resolution data.…”

Section: Discussionmentioning

confidence: 99%

“…However, with the increasing use of artificial intelligence (AI) and machine learning algorithms, there is now a great need for precise phenotype-to-genotype measurements to better understand the sequence space of the respective protein. The resulting high-quality datasets can then be used to train computer-based models with the aim to improve accuracy and insight of subsequent predictions (9).…”

Section: Introductionmentioning

confidence: 99%

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Heat pre-treatment reduces multiplicity of plasmid transformations in yeast during electroporation, without diminishing the transformation efficiency

Wäneskog,

Hoch-Schneider,

Garg

et al. 2024

Preprint

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High-throughput DNA transformation techniques are invaluable when creating high-diversity mutant libraries, and the success rate of any protein engineering endeavors is directly dependent on both the size and diversity of the mutant library that is to be screened. It is also widely accepted that in both bacteria and yeast there is an inverse correlation between the DNA transformation efficiency and the likelihood of transforming multiple DNA molecules into each cell. However, most successful high-throughput mutant screening efforts require high quality libraries, i.e., libraries comprised of cells with a clear phenotype-to-genotype relationship (one genotype/cell). Thus, DNA transformation methods with a high multiplicity of transformation are highly undesirable and detrimental to most mutant screening assays. Here we describe a simple, robust, and highly efficient yeast plasmid DNA transformation methodology, using a dual heat-shock and electroporation approach (HEEL) that generates more than 2 x 107plasmid-transformed yeast cells per reaction, while simultaneously increasing the fraction of mono-transformed cells from 20% to more than 70% of the transformed population. By also using an automated yeast genotyping workflow coupled with a dual-barcoding approach, consisting of a SNP and high-diversity barcode (10N), we can consistently identify and enumerate unique plasmid genotypes within a heterogeneous population merely through Sanger sequencing. We demonstrate here that the size and quality of a transformed library no longer need to be inversely correlated when transforming large mutant DNA libraries in yeast using highly efficient DNA electroporation methods.SignificanceWith the recent expansion of artificial intelligence in the field of synthetic biology there has never been a greater need for high-quality data and reliable measurements of phenotype-to-genotype relationships. However, one major obstacle to creating accurate computer-based models is the current abundance of low-quality phenotypic measurements originating from numerous high-throughput, but low-resolution assays. Rather than increasing the quantity of measurements, new studies should aim to generate as accurate measurements as possible. The HEEL methodology presented here aims to address this issue by minimizing the problem of multi-plasmid uptake during high-throughput yeast DNA transformations, which leads to the creation of heterogeneous cellular genotypes. HEEL should enable highly accurate phenotype-to-genotype measurements going forward, which could be used to construct better computer-based models.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Heat pre-treatment reduces multiplicity of plasmid transformations in yeast during electroporation, without diminishing the transformation efficiency

Wäneskog,

Hoch-Schneider,

Garg

et al. 2024

Preprint

Self Cite

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“…Although engineered cell contact-based signalling has been widely demonstrated in mammalian cell systems, it has not been exploited for use in yeast, despite yeast being important for high-throughput research in investigating biological mechanisms and for drug development 15,16 . In past work, yeast surface display technologies have been used to establish cell contact-based signalling associations directly between yeast and mammalian cells to explore immune-related protein interactions 17,18 .…”

Section: Introductionmentioning

confidence: 99%

Engineered yeast multicellularity via synthetic cell-cell adhesion and direct-contact signalling

Meng,

Shaw,

Kam

et al. 2024

Preprint

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Coordination of behaviour in multicellular systems is one the main ways that nature increases the complexity of biological function in organisms and communities. WhileSaccharomyces cerevisiaeyeast is used extensively in research and biotechnology, it is a unicellular organism capable of only limited multicellular states. Here we expand the possibilities for engineering multicellular behaviours in yeast by developing modular toolkits for two key mechanisms seen in multicellularity, contact-dependent signalling and specific cell-to-cell adhesion. MARS (Mating-peptideAnchoredResponseSystem) is a toolkit based on surface-displayed fungal mating peptides and G protein-coupled receptor (GPCR) signalling which can mimic juxtacrine signalling between yeasts. SATURN (SaccharomycesAdhesionToolkit for multicellUlar patteRNing) surface displays adhesion-proteins pairs on yeasts and facilitates the creation of cell aggregation patterns. Together they can be used to create multicellular logic circuits, equivalent to developmental programs that lead to cell differentiation based on the local population. Using MARS and SATURN, we further developed JUPITER (JUxtacrine sensor forProtein-protein InTERaction), a genetic sensor for assaying protein-protein interactions in culture, demonstrating this as a tool to select for high affinity binders among a population of mutated nanobodies. Collectively, MARS, SATURN, and JUPITER present valuable tools that facilitate the engineering of complex multicellularity with yeast and expand the scope of its biotechnological applications.

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High‐throughput G protein‐coupled receptor‐based autocrine screening for secondary metabolite production in yeast

Saleski,

Peng,

Lengger

et al. 2024

Biotech & Bioengineering

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Biosensors are valuable tools in accelerating the test phase of the design‐build‐test‐learn cycle of cell factory development, as well as in bioprocess monitoring and control. G protein‐coupled receptor (GPCR)‐based biosensors enable cells to sense a wide array of molecules and environmental conditions in a specific manner. Due to the extracellular nature of their sensing, GPCR‐based biosensors require compartmentalization of distinct genotypes when screening production levels of a strain library to ensure that detected levels originate exclusively from the strain under assessment. Here, we explore the integration of production and sensing modalities into a single Saccharomyces cerevisiae strain and compartmentalization using three different methods: (1) cultivation in microtiter plates, (2) spatial separation on agar plates, and (3) encapsulation in water‐in‐oil‐in‐water double emulsion droplets, combined with analysis and sorting via a fluorescence‐activated cell sorting machine. Employing tryptamine and serotonin as proof‐of‐concept target molecules, we optimize biosensing conditions and demonstrate the ability of the autocrine screening method to enrich for high producers, showing the enrichment of a serotonin‐producing strain over a nonproducing strain. These findings illustrate a workflow that can be adapted to screening for a wide range of complex chemistry at high throughput using commercially available microfluidic systems.

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Yeast-based screening platforms to understand and improve human health

Cited by 5 publications

References 138 publications

Heat pre-treatment reduces multiplicity of plasmid transformations in yeast during electroporation, without diminishing the transformation efficiency

Heat pre-treatment reduces multiplicity of plasmid transformations in yeast during electroporation, without diminishing the transformation efficiency

Engineered yeast multicellularity via synthetic cell-cell adhesion and direct-contact signalling

High‐throughput G protein‐coupled receptor‐based autocrine screening for secondary metabolite production in yeast

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