Tailoring baker's yeast Saccharomyces cerevisiae for functional testing of channelrhodopsin.

Channelrhodopsin 2 (ChR2) and its variants are the most frequent tools for remote manipulation of electrical properties in cells via light. Ongoing attempts try to enlarge their functional spectrum with respect to ion selectivity, light sensitivity and protein trafficking by mutations, protein engineering and environmental mining of ChR2 variants. A shortcoming in the required functional testing of large numbers of ChR2 variants is the lack of an easy screening system. Baker's yeast, which was successfully employed for testing ion channels from eukaryotes has not yet been used for screening of ChR2s, because they neither produce the retinal chromophore nor its precursor carotenoids. We found that addition of retinal to the external medium was not sufficient for detecting robust ChR activity in yeast in simple growth assays. This obstacle was overcome by metabolic engineering of a yeast strain, which constitutively produces retinal. In proof of concept experiments we functionally express different ChR variants in these cells and monitor their blue light induced activity in simple growth assays. We find that light activation of ChR augments an influx of Na+ with a consequent inhibition of cell growth. In a K+ uptake deficient yeast strain, growth can be rescued in selective medium by the blue light induced K+ conductance of ChR. This yeast strain can now be used as chassis for screening of new functional ChR variants and mutant libraries in simple yeast growth assays under defined selective conditions.

A MAD7-based genome editing system for Escherichia coli.

A broad variety of biomolecules is industrially produced in bacteria and yeasts. These microbial expression hosts can be optimized through genetic engineering using CRISPR tools. Here, we designed and characterized such a modular genome editing system based on the Cas12a-like RNA-guided nuclease MAD7 in Escherichia coli. This system enables the efficient generation of single nucleotide polymorphisms (SNPs) or gene deletions and can directly be used with donor DNA from benchtop DNA assembly to increase throughput. We combined multiple edits to engineer an E. coli strain with reduced overflow metabolism and increased plasmid yield, highlighting the versatility and industrial applicability of this approach.

Novel auto-selection systems for transformation selection of Saccharomyces cerevisiae in rich complex media.

The most widely used strategy for selection of yeast transformed with episomal plasmids comprises the use of auxotrophic yeast strains in combination with vectors containing complementing prototrophic marker genes. Another approach uses heterologous genes or cassettes which, if present in the vector, render the otherwise sensitive yeast strain resistant to antibiotics. In addition, auto-selection systems for Saccharomyces cerevisiae have been developed that eliminate the requirement for synthetic drop-out media or the use of antibiotics for transformation selection and subsequent plasmid maintenance in expression cultures. Here we describe a combination of host strain and vector system introducing a novel concept of auto-selection systems that allows for easy and robust propagation of host cells deleted in essential genes in supplemented media before being transformed with rescuing plasmids. With that, our approach is favorable over commonly used selection strategies and has major advantage over other auto-selection systems. Our approach complements the auto-selection toolbox already available for S. cerevisiae, thus contributing a novel system that enables the use of complex peptone-based media for protein expression and metabolic engineering approaches. We therefore expect that this new strategy will be of general interest to the yeast research community in academia and industry.

Lipid determinants of endocytosis and exocytosis in budding yeast.

Cells maintain physicochemical characteristics of membranes in order to allow for proper function of membrane-associated cellular processes, such as endocytosis and exocytosis. To investigate the interplay between membrane properties and biological processes, we applied lipid engineering approaches that allowed for systematic manipulation of fatty acid unsaturation and sterol biosynthesis, the main regulators of membrane fluidity. In combination with electrophysiological membrane capacitance measurements, we were able to study the dependence of the endo- and exocytic activity of Saccharomyces cerevisiae on membrane lipid composition in vivo. We found that a strong decrease in the cell's total ergosterol content leads to a severely reduced frequency of vesicle fission (endocytosis), whereas the exocytic activity remained largely unaffected. In contrast, increased lipid saturation lowered both endocytic and the exocytic activity, with the former being more severely affected. We were able to correlate the decreased ratio of endocytic/exocytic frequencies (fendo/fexo) upon lipid perturbation with the growth of yeast protoplasts, which is based on a surface enlargement resulting from a net excess of exocytic over endocytic flux. Experiments using clathrin-deficient mutants confirm a correlation between reduced endocytic activity and increased size of intact walled cells, as well as accelerated protoplast growth. These data show that lipid composition is intimately tied to membrane trafficking in yeast cells and suggest that endocytosis is particularly dependent on the lipid-defined properties of cell membrane.

Preloading budding yeast with all-in-one CRISPR/Cas9 vectors for easy and high-efficient genome editing.

The CRISPR/Cas9 technology has greatly improved genome editing in Saccharomyces cerevisiae over recent years. However, several current CRISPR/Cas9 systems suffer from work-intensive cloning procedures and/or the requirement of co-transforming target cells with multiple system components simultaneously which can reduce the effectivity of such applications. Here, we present a new set of all-in-one CRISPR/Cas9 vectors that combine unique benefits of different already existent systems in order to further expand the technology's design possibilities. Our vectors mediate constitutive gRNA expression whereas Cas9 expression is either driven from a constitutive or an inducible promoter. The introduction of desired gRNA targeting sequences into our inducible single gRNA vector relies just on in vivo homologous recombination-mediated assembly of overlapping single-stranded oligonucleotides, thus reducing efforts of plasmid cloning to an absolute minimum. By employing the inducible system, yeast cells can be easily preloaded with plasmids encoding for a functional CRISPR/Cas9 system, thereby chronologically separating the cloning procedure from the genome editing step. Gene knockouts could be achieved with high efficiency and effectivity by simply transforming preloaded cells with a selectable disruption cassette without the need of co-introducing any CRISPR/Cas9 system component. We also show the feasibility of efficient gene knockouts even when multiple gene copies were present such as in non-haploid strain backgrounds as well as the simultaneous deletion of two different genes in a haploid genetic background by using a multiplex variant of our inducible vector. The versatile applicability of our inducible vector system was further demonstrated by CRISPR/Cas9-mediated mating type switching of yeast.

Vesicle fusion and fission in plants and yeast.

Measurements of the membrane capacitance on animal cells has provided an excellent technique for monitoring of exo- and endocytotic activity in intact living cells. Here we review recent data in which the same technique was applied to plant cells and cells of the budding yeast Saccharomyces cerevisiae. The data show that unitary exo- and endocytotic events can also be measured with the same technique after removing the cell wall from these cells. The resulting protoplasts execute the same type of transient and permanent fusion/fission that is known from animal cells. Also the size of the vesicles, which are fusing or budding, are of the same order of magnitude as those recorded in animal cells. Together these data support the view of an evolutionary conserved mechanism for unitary exo- and endocytosis events in eukaryotes. The successful recordings of exo- and endocytotic activity in Saccharomyces cerevisiae by capacitance measurements now pave the way for correlating the abundant information on the molecular machinery of exo- and endocytosis in this model organism with distinct functional properties.

Lipid engineering reveals regulatory roles for membrane fluidity in yeast flocculation and oxygen-limited growth.

Cells modulate lipid metabolism in order to maintain membrane homeostasis. Here we use a metabolic engineering approach to manipulate the stoichiometry of fatty acid unsaturation, a regulator of cell membrane fluidity, in Saccharomyces cerevisiae. Unexpectedly, reduced lipid unsaturation triggered cell-cell adhesion (flocculation), a phenomenon characteristic of industrial yeast but uncommon in laboratory strains. We find that ER lipid saturation sensors induce expression of FLO1 - encoding a cell wall polysaccharide binding protein - independently of its canonical regulator. In wild-type cells, Flo1p-dependent flocculation occurs under oxygen-limited growth, which reduces unsaturated lipid synthesis and thus serves as the environmental trigger for flocculation. Transcriptional analysis shows that FLO1 is one of the most highly induced genes in response to changes in lipid unsaturation, and that the set of membrane fluidity-sensitive genes is globally activated as part of the cell's long-term response to hypoxia during fermentation. Our results show how the lipid homeostasis machinery of budding yeast is adapted to carry out a broad response to an environmental stimulus important in biotechnology.