Killer yeasts: expanding frontiers in the age of synthetic biology.

Killer yeasts secrete protein toxins that are selectively lethal to other yeast and filamentous fungi. These exhibit exceptional genetic and functional diversity, and have several biotechnological applications. However, despite decades of research, several limitations hinder their widespread adoption. In this perspective we contend that technical advances in synthetic biology present an unprecedented opportunity to unlock the full potential of yeast killer systems across a spectrum of applications. By leveraging these new technologies, engineered killer toxins may emerge as a pivotal new tool to address antifungal resistance and food security. Finally, we speculate on the biotechnological potential of re-engineering host double-stranded (ds) RNA mycoviruses, from which many toxins derive, as a safe and noninfectious system to produce designer RNA.

Improving homology-directed repair by small molecule agents for genetic engineering in unconventional yeast?-Learning from the engineering of mammalian systems.

The ability to precisely edit genomes by deleting or adding genetic information enables the study of biological functions and the building of efficient cell factories. In many unconventional yeasts, such as those promising new hosts for cell factory design but also human pathogenic yeasts and food spoilers, this progress has been limited by the fact that most yeasts favour non-homologous end joining (NHEJ) over homologous recombination (HR) as a DNA repair mechanism, impairing genetic access to these hosts. In mammalian cells, small molecules that either inhibit proteins involved in NHEJ, enhance protein function in HR, or arrest the cell cycle in HR-dominant phases are regarded as promising agents for the simple and transient increase of HR-mediated genome editing without the need for a priori host engineering. Only a few of these chemicals have been applied to the engineering of yeast, although the targeted proteins are mostly conserved, making chemical agents a yet-underexplored area for enhancing yeast engineering. Here, we consolidate knowledge of the available small molecules that have been used to improve HR efficiency in mammalian cells and the few ones that have been used in yeast. We include available high-throughput-compatible NHEJ/HR quantification assays that could be used to screen for and isolate yeast-specific inhibitors.

Ten simple rules for managing laboratory information.

Information is the cornerstone of research, from experimental (meta)data and computational processes to complex inventories of reagents and equipment. These 10 simple rules discuss best practices for leveraging laboratory information management systems to transform this large information load into useful scientific findings.

A Modular Cloning Toolkit Including CRISPRi for the Engineering of the Human Fungal Pathogen and Biotechnology Host Candida glabrata.

The yeast Candida glabrata is an emerging, often drug-resistant opportunistic human pathogen that can cause severe systemic infections in immunocompromised individuals. At the same time, it is a valuable biotechnology host that naturally accumulates high levels of pyruvate─a valuable chemical precursor. Tools for the facile engineering of this yeast could greatly accelerate studies on its pathogenicity and its optimization for biotechnology. While a few tools for plasmid-based expression and genome engineering have been developed, there is no well-characterized cloning toolkit that would allow the modular assembly of pathways or genetic circuits. Here, by characterizing the Saccharomyces cerevisiae-based yeast molecular cloning toolkit (YTK) in C. glabrata and by adding missing components, we build a well-characterized CgTK (C. glabrata toolkit). We used the CgTK to build a CRISPR interference system for C. glabrata that can be used to generate selectable phenotypes via single-gRNA targeting such as is required for genome-wide library screens.

Functional Synthetic Biology.

Synthetic biologists have made great progress over the past decade in developing methods for modular assembly of genetic sequences and in engineering biological systems with a wide variety of functions in various contexts and organisms. However, current paradigms in the field entangle sequence and functionality in a manner that makes abstraction difficult, reduces engineering flexibility and impairs predictability and design reuse. Functional Synthetic Biology aims to overcome these impediments by focusing the design of biological systems on function, rather than on sequence. This reorientation will decouple the engineering of biological devices from the specifics of how those devices are put to use, requiring both conceptual and organizational change, as well as supporting software tooling. Realizing this vision of Functional Synthetic Biology will allow more flexibility in how devices are used, more opportunity for reuse of devices and data, improvements in predictability and reductions in technical risk and cost.

Multicolor plate reader fluorescence calibration.

Plate readers are commonly used to measure cell growth and fluorescence, yet the utility and reproducibility of plate reader data is limited by the fact that it is typically reported in arbitrary or relative units. We have previously established a robust serial dilution protocol for calibration of plate reader measurements of absorbance to estimated bacterial cell count and for green fluorescence from proteins expressed in bacterial cells to molecules of equivalent fluorescein. We now extend these protocols to calibration of red fluorescence to the sulforhodamine-101 fluorescent dye and blue fluorescence to Cascade Blue. Evaluating calibration efficacy via an interlaboratory study, we find that these calibrants do indeed provide comparable precision to the prior calibrants and that they enable effective cross-laboratory comparison of measurements of red and blue fluorescence from proteins expressed in bacterial cells.

Peptide-Dependent Growth in Yeast via Fine-Tuned Peptide/GPCR-Activated Essential Gene Expression.

Building multicellular microbial consortia that communicate with each other and perform programmed functionalities is the next milestone for synthetic biology. Achieving cell-cell communication within these communities requires programming of the transduction of an extracellular signal into a customized intracellular response. G-protein-coupled receptors (GPCRs) are attractive candidates for engineering signal transduction as they can sense extracellular events with high sensitivity and specificity and transduce them into complex intracellular programs. We recently developed a scalable cell-cell communication language based on fungal mating GPCRs and their secreted peptide ligands. This language allows the assembly of engineered yeast strains into multicellular communication networks and allows them to be made interdependent by peptide signaling. In peptide signaling, one cell secretes a peptide that supports the growth of another cell at nanomolar concentrations, a scalable approach for engineering interdependence. Here we address the challenge of correlating the doubling time of Saccharomyces cerevisiae cells with an increasing external peptide concentration by linking GPCR activation to the expression of an essential gene. The required fine-tuning of downstream signaling is achieved via the transcriptional titration of a set of orthogonal GPCR-activated transcription factors, a series of corresponding promoters with different output dynamics, and the use of chemically recoded peptide ligands with varying activation potentials. As such, our work establishes three control points that allow the tuning of the basal and maximal activation of the GPCR response, fold change activation, and response sensitivity. The presented results enable the implementation of peptide-dependent and peptide-tunable growth but could also facilitate the design and calibration of more complex GPCR-controlled synthetic functionality in the future.

Oligo Pools as an Affordable Source of Synthetic DNA for Cost-Effective Library Construction in Protein- and Metabolic Pathway Engineering.

The construction of custom libraries is critical for rational protein engineering and directed evolution. Array-synthesized oligo pools of thousands of user-defined sequences (up to ∼350 bases in length) have emerged as a low-cost commercially available source of DNA. These pools cost ≤10 % (depending on error rate and length) of other commercial sources of custom DNA, and this significant cost difference can determine whether an enzyme engineering project can be realized on a given research budget. However, while being cheap, oligo pools do suffer from a low concentration of individual oligos and relatively high error rates. Several powerful techniques that specifically make use of oligo pools have been developed and proven valuable or even essential for next-generation protein and pathway engineering strategies, such as sequence-function mapping, enzyme minimization, or de-novo design. Here we consolidate the knowledge on these techniques and their applications to facilitate the use of oligo pools within the protein engineering community.

A buffered media system for yeast batch culture growth.

BACKGROUND: Fungi are premier hosts for the high-yield secretion of proteins for biomedical and industrial applications. The stability and activity of these secreted proteins is often dependent on the culture pH. As yeast acidifies the commonly used synthetic complete drop-out (SD) media that contains ammonium sulfate, the pH of the media needs to be buffered in order to maintain a desired extracellular pH during biomass production. At the same time, many buffering agents affect growth at the concentrations needed to support a stable pH. Although the standard for biotechnological research and development is shaken batch cultures or microtiter plate cultures that cannot be easily automatically pH-adjusted during growth, there is no comparative study that evaluates the buffering capacity and growth effects of different media types across pH-values in order to develop a pH-stable batch culture system. RESULTS: We systematically test the buffering capacity and growth effects of a citrate-phosphate buffer (CPB) from acidic to neutral pH across different media types. These media types differ in their nitrogen source (ammonium sulfate, urea or both). We find that the widely used synthetic drop-out media that uses ammonium sulfate as nitrogen source can only be effectively buffered at buffer concentrations that also affect growth. At lower concentrations, yeast biomass production still acidifies the media. When replacing the ammonium sulfate with urea, the media alkalizes. We then develop a medium combining ammonium sulfate and urea which can be buffered at low CPB concentrations that do not affect growth. In addition, we show that a buffer based on Tris/HCl is not effective in maintaining any of our media types at neutral pH even at relatively high concentrations. CONCLUSION: Here we show that the buffering of yeast batch cultures is not straight-forward and addition of a buffering agent to set a desired starting pH does not guarantee pH-maintenance during growth. In response, we present a buffered media system based on an ammonium sulfate/urea medium that enables relatively stable pH-maintenance across a wide pH-range without affecting growth. This buffering system is useful for protein-secretion-screenings, antifungal activity assays, as well as for other pH-dependent basic biology or biotechnology projects.

Author Correction: A scalable peptide-GPCR language for engineering multicellular communication.

The original version of this Article omitted a declaration from the Competing Interests statement, which should have included the following: 'J.D.B. is a founder and Director of the following: Neochromosome, Inc., the Center of Excellence for Engineering Biology, and CDI Labs, Inc. and serves on the Scientific Advisory Board of the following: Modern Meadow, Inc., Recombinetics, Inc., and Sample6, Inc.'. This has now been corrected in both the PDF and HTML versions of the Article.

A scalable peptide-GPCR language for engineering multicellular communication.

Engineering multicellularity is one of the next breakthroughs for Synthetic Biology. A key bottleneck to building multicellular systems is the lack of a scalable signaling language with a large number of interfaces that can be used simultaneously. Here, we present a modular, scalable, intercellular signaling language in yeast based on fungal mating peptide/G-protein-coupled receptor (GPCR) pairs harnessed from nature. First, through genome-mining, we assemble 32 functional peptide-GPCR signaling interfaces with a range of dose-response characteristics. Next, we demonstrate that these interfaces can be combined into two-cell communication links, which serve as assembly units for higher-order communication topologies. Finally, we show 56 functional, two-cell links, which we use to assemble three- to six-member communication topologies and a three-member interdependent community. Importantly, our peptide-GPCR language is scalable and tunable by genetic encoding, requires minimal component engineering, and should be massively scalable by further application of our genome mining pipeline or directed evolution.

Sequence-based prediction of permissive stretches for internal protein tagging and knockdown.

BACKGROUND: Internal tagging of proteins by inserting small functional peptides into surface accessible permissive sites has proven to be an indispensable tool for basic and applied science. Permissive sites are typically identified by transposon mutagenesis on a case-by-case basis, limiting scalability and their exploitation as a system-wide protein engineering tool. METHODS: We developed an apporach for predicting permissive stretches (PSs) in proteins based on the identification of length-variable regions (regions containing indels) in homologous proteins. RESULTS: We verify that a protein's primary structure information alone is sufficient to identify PSs. Identified PSs are predicted to be predominantly surface accessible; hence, the position of inserted peptides is likely suitable for diverse applications. We demonstrate the viability of this approach by inserting a Tobacco etch virus protease recognition site (TEV-tag) into several PSs in a wide range of proteins, from small monomeric enzymes (adenylate kinase) to large multi-subunit molecular machines (ATP synthase) and verify their functionality after insertion. We apply this method to engineer conditional protein knockdowns directly in the Escherichia coli chromosome and generate a cell-free platform with enhanced nucleotide stability. CONCLUSIONS: Functional internally tagged proteins can be rationally designed and directly chromosomally implemented. Critical for the successful design of protein knockdowns was the incorporation of surface accessibility and secondary structure predictions, as well as the design of an improved TEV-tag that enables efficient hydrolysis when inserted into the middle of a protein. This versatile and portable approach can likely be adapted for other applications, and broadly adopted. We provide guidelines for the design of internally tagged proteins in order to empower scientists with little or no protein engineering expertise to internally tag their target proteins.

A modular yeast biosensor for low-cost point-of-care pathogen detection.

The availability of simple, specific, and inexpensive on-site detection methods is of key importance for deployment of pathogen surveillance networks. We developed a nontechnical and highly specific colorimetric assay for detection of pathogen-derived peptides based on Saccharomyces cerevisiae-a genetically tractable model organism and household product. Integrating G protein-coupled receptors with a visible, reagent-free lycopene readout, we demonstrate differential detection of major human, plant, and food fungal pathogens with nanomolar sensitivity. We further optimized a one-step rapid dipstick prototype that can be used in complex samples, including blood, urine, and soil. This modular biosensor can be economically produced at large scale, is not reliant on cold-chain storage, can be detected without additional equipment, and is thus a compelling platform scalable to global surveillance of pathogens.