Peptide Variant Detection by a Living Yeast Biosensor via an Epitope-Selective Protease.

We previously demonstrated that we could hijack the fungal pheromone signaling pathway to provide a living yeast biosensor where peptide biomarkers were recognized by G-protein-coupled receptors and engineered to transcribe a readout. Here, we demonstrated that the protease could be reintroduced to the biosensor to provide a simple mechanism for distinguishing single-amino-acid changes in peptide ligands that, otherwise, would likely be difficult to detect using binding-based assays. We characterized the dose-response curves for five fungal pheromone G-protein-coupled receptors, peptides, and proteases-Saccharomyces cerevisiae, Candida albicans, Schizosaccharomyces pombe, Schizosaccharomyces octosporus, and Schizosaccharomyces japonicus. Alanine scanning was carried out for the most selective of these-S. cerevisiae and C. albicans-with and without the protease. Two peptide variants were discovered, which showed diminished cleavage by the protease (CaPep2A and CaPep2A13A). Those peptides were then distinguished by utilizing the biosensor strains with and without the protease, which selectively cleaved and altered the apparent concentration of peptide required for half-maximal activation for 2 peptides-CaPep and CaPep13A, respectively-by more than one order of magnitude. These results support the hypothesis that the living yeast biosensor with a sequence-specific protease can translate single-amino-acid changes into more than one order of magnitude apparent shift in the concentration of peptide required for half-maximal activation. With further engineering by computational modeling and directed evolution, the biosensor could likely distinguish a wide variety of peptide sequences beyond the alanine scanning carried out here. In the future, we envision incorporating proteases into our living yeast biosensor for use as a point of care diagnostic, a scalable communication language, and other applications.

High-Titer Production of the Fungal Anhydrotetracycline, TAN-1612, in Engineered Yeasts.

Antibiotic resistance is a growing global health threat, demanding urgent responses. Tetracyclines, a widely used antibiotic class, are increasingly succumbing to antibiotic resistance; generating novel analogues is therefore a top priority for public health. Fungal tetracyclines provide structural and enzymatic diversity for novel tetracycline analogue production in tractable heterologous hosts, like yeasts, to combat antibiotic-resistant pathogens. Here, we successfully engineered Saccharomyces cerevisiae (baker's yeast) and Saccharomyces boulardii (probiotic yeast) to produce the nonantibiotic fungal anhydrotetracycline, TAN-1612, in synthetic defined media─necessary for clean purifications─through heterologously expressing TAN-1612 genes mined from the fungus, Aspergillus niger ATCC 1015. This was accomplished via (i) a promoter library-based combinatorial pathway optimization of the biosynthetic TAN-1612 genes coexpressed with a putative TAN-1612 efflux pump, reducing TAN-1612 toxicity in yeasts while simultaneously increasing supernatant titers and (ii) the development of a medium-throughput UV-visible spectrophotometric assay that facilitates TAN-1612 combinatorial library screening. Through this multipronged approach, we optimized TAN-1612 production, yielding an over 450-fold increase compared to previously reported S. cerevisiae yields. TAN-1612 is an important tetracycline analogue precursor, and we thus present the first step toward generating novel tetracycline analogue therapeutics to combat current and emerging antibiotic resistance. We also report the first heterologous production of a fungal polyketide, like TAN-1612, in the probiotic S. boulardii. This highlights that engineered S. boulardii can biosynthesize complex natural products like tetracyclines, setting the stage to equip probiotic yeasts with synthetic therapeutic functionalities to generate living therapeutics or biocontrol agents for clinical and agricultural applications.

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.

Synthetic biology: at the crossroads of genetic engineering and human therapeutics-a Keystone Symposia report.

Synthetic biology has the potential to transform cell- and gene-based therapies for a variety of diseases. Sophisticated tools are now available for both eukaryotic and prokaryotic cells to engineer cells to selectively achieve therapeutic effects in response to one or more disease-related signals, thus sparing healthy tissue from potentially cytotoxic effects. This report summarizes the Keystone eSymposium "Synthetic Biology: At the Crossroads of Genetic Engineering and Human Therapeutics," which took place on May 3 and 4, 2021. Given that several therapies engineered using synthetic biology have entered clinical trials, there was a clear need for a synthetic biology symposium that emphasizes the therapeutic applications of synthetic biology as opposed to the technical aspects. Presenters discussed the use of synthetic biology to improve T cell, gene, and viral therapies, to engineer probiotics, and to expand upon existing modalities and functions of cell-based therapies.

Heterologous Catalysis of the Final Steps of Tetracycline Biosynthesis by Saccharomyces cerevisiae.

Developing treatments for antibiotic resistant bacterial infections is among the highest priority public health challenges worldwide. Tetracyclines, one of the most important classes of antibiotics, have fallen prey to antibiotic resistance, necessitating the generation of new analogs. Many tetracycline analogs have been accessed through both total synthesis and semisynthesis, but key C-ring tetracycline analogs remain inaccessible. New methods are needed to unlock access to these analogs, and heterologous biosynthesis in a tractable host such as Saccharomyces cerevisiae is a candidate method. C-ring analog biosynthesis can mimic nature's biosynthesis of tetracyclines from anhydrotetracyclines, but challenges exist, including the absence of the unique cofactor F420 in common heterologous hosts. Toward this goal, this paper describes the biosynthesis of tetracycline from anhydrotetracycline in S. cerevisiae heterologously expressing three enzymes from three bacterial hosts: the anhydrotetracycline hydroxylase OxyS, the dehydrotetracycline reductase CtcM, and the F420 reductase FNO. This biosynthesis of tetracycline is enabled by OxyS performing just one hydroxylation step in S. cerevisiae despite its previous characterization as a double hydroxylase. This single hydroxylation enabled us to purify and structurally characterize a hypothetical intermediate in oxytetracycline biosynthesis that can explain structural differences between oxytetracycline and chlortetracycline. We show that Fo, a synthetically accessible derivative of cofactor F420, can replace F420 in tetracycline biosynthesis. Critically, the use of S. cerevisiae for the final steps of tetracycline biosynthesis described herein sets the stage to achieve a total biosynthesis of tetracycline as well as novel tetracycline analogs in S. cerevisiae with the potential to combat antibiotic-resistant bacteria.

Genetic Code Expansion: A Brief History and Perspective.

Since the establishment of site-specific mutagenesis of single amino acids to interrogate protein function in the 1970s, biochemists have sought to tailor protein structure in the native cell environment. Fine-tuning the chemical properties of proteins is an indispensable way to address fundamental mechanistic questions. Unnatural amino acids (UAAs) offer the possibility to expand beyond the 20 naturally occurring amino acids in most species and install new and useful chemical functions. Here, we review the literature about advances in UAA incorporation technology from chemoenzymatic aminoacylation of modified tRNAs to in vitro translation systems to genetic encoding of UAAs in the native cell environment and whole organisms. We discuss innovative applications of the UAA technology to challenges in bioengineering and medicine.

Draft Genome Sequence of Saccharomyces cerevisiae LW2591Y, a Laboratory Strain for In Vivo Multigene Assemblies.

Saccharomyces cerevisiae is an industrially preferred cell factory for the heterologous production of proteins and chemicals. Here, we present the draft genome sequence of the laboratory strain Saccharomyces cerevisiae LW2591Y, which has been designed for robust and efficient assembly of multigene pathways.

Detection of Nav1.5 Conformational Change in Mammalian Cells Using the Noncanonical Amino Acid ANAP.

Nav1.5 inactivation is necessary for healthy conduction of the cardiac action potential. Genetic mutations of Nav1.5 perturb inactivation and cause potentially fatal arrhythmias associated with long QT syndrome type 3. The exact structural dynamics of the inactivation complex is unknown. To sense inactivation gate conformational change in live mammalian cells, we incorporated the solvatochromic fluorescent noncanonical amino acid 3-((6-acetylnaphthalen-2-yl)amino)-2-aminopropanoic acid (ANAP) into single sites in the Nav1.5 inactivation gate. ANAP was incorporated in full-length and C-terminally truncated Nav1.5 channels using mammalian cell synthetase-tRNA technology. ANAP-incorporated channels were expressed in mammalian cells, and they exhibited pathophysiological function. A spectral imaging potassium depolarization assay was designed to detect ANAP emission shifts associated with Nav1.5 conformational change. Site-specific intracellular ANAP incorporation affords live-cell imaging and detection of Nav1.5 inactivation gate conformational change in mammalian cells.

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.

Interrogation of Eukaryotic Stop Codon Readthrough Signals by in Vitro RNA Selection.

RNA signals located downstream of stop codons in eukaryotic mRNAs can stimulate high levels of translational readthrough by the ribosome, thereby giving rise to functionally distinct C-terminally extended protein products. Although many readthrough events have been previously discovered in Nature, a broader description of the stimulatory RNA signals would help to identify new reprogramming events in eukaryotic genes and provide insights into the molecular mechanisms of readthrough. Here, we explore the RNA reprogramming landscape by performing in vitro translation selections to enrich RNA readthrough signals de novo from a starting randomized library comprising >1013 unique sequence variants. Selection products were characterized using high-throughput sequencing, from which we identified primary sequence and secondary structure readthrough features. The activities of readthrough signals, including three novel sequence motifs, were confirmed in cellular reporter assays. Then, we used machine learning and our HTS data to predict readthrough activity from human 3'-untranslated region sequences. This led to the discovery of >1.5% readthrough in four human genes (CDKN2B, LEPROTL1, PVRL3, and SFTA2). Together, our results provide valuable insights into RNA-mediated translation reprogramming, offer tools for readthrough discovery in eukaryotic genes, and present new opportunities to explore the biological consequences of stop codon readthrough in humans.

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.

d-Amino Acid-Mediated Translation Arrest Is Modulated by the Identity of the Incoming Aminoacyl-tRNA.

A complete understanding of the determinants that restrict d-amino acid incorporation by the ribosome, which is of interest to both basic biologists and the protein engineering community, remains elusive. Previously, we demonstrated that d-amino acids are successfully incorporated into the C-terminus of the nascent polypeptide chain. Ribosomes carrying the resulting peptidyl-d-aminoacyl-tRNA (peptidyl-d-aa-tRNA) donor substrate, however, partition into subpopulations that either undergo translation arrest through inactivation of the ribosomal peptidyl-transferase center (PTC) or remain translationally competent. The proportion of each subpopulation is determined by the identity of the d-amino acid side chain. Here, we demonstrate that the identity of the aminoacyl-tRNA (aa-tRNA) acceptor substrate that is delivered to ribosomes carrying a peptidyl-d-aa-tRNA donor further modulates this partitioning. Our discovery demonstrates that it is the pairing of the peptidyl-d-aa-tRNA donor and the aa-tRNA acceptor that determines the activity of the PTC. Moreover, we provide evidence that both the amino acid and tRNA components of the aa-tRNA acceptor contribute synergistically to the extent of arrest. The results of this work deepen our understanding of the mechanism of d-amino acid-mediated translation arrest and how cells avoid this precarious obstacle, reveal similarities to other translation arrest mechanisms involving the PTC, and provide a new route for improving the yields of engineered proteins containing d-amino acids.

A Yeast Three Hybrid Assay for Metabolic Engineering of Tetracycline Derivatives.

Metabolic engineering stands to transform the discovery and production of a wide range of chemicals, but metabolic engineering currently demands considerable resource investments that restrict commercial application. To facilitate the applicability of metabolic engineering, general high-throughput and readily implemented technologies are needed to assay vast libraries of strains producing desirable chemicals. Toward this end, we describe here the development of a yeast three hybrid (Y3H) assay as a general, high-throughput, versatile and readily implemented approach for the detection of target molecule biosynthesis. Our system detects target molecule biosynthesis through a change in reporter gene transcription that results from the binding of the target molecule to a modular protein receptor. We demonstrate the use of the Y3H assay for detecting the biosynthesis of tetracyclines, a major class of antibiotics, based on the interaction between tetracyclines and the tetracycline repressor protein (TetR). Various tetracycline derivatives can be detected using our assay, whose versatility enables its use both as a screen and a selection to match the needs and instrumentation of a wide range of end users. We demonstrate the applicability of the Y3H assay to metabolic engineering by differentiating between producer and nonproducer strains of the natural product tetracycline TAN-1612. The Y3H assay is superior to state-of-the-art HPLC-MS methods in throughput and limit of detection of tetracycline derivatives. Finally, our establishment of the Y3H assay for detecting the biosynthesis of a tetracycline supports the generality of the Y3H assay for detecting the biosynthesis of many other target molecules.

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.

Fluorescence Polarization Assay for Small Molecule Screening of FK506 Biosynthesized in 96-Well Microtiter Plates.

The fluorescence polarization (FP) assay has been widely used to study enzyme kinetics, antibody-antigen interactions, and other biological interactions. We propose that the FP assay can be adapted as a high-throughput and potentially widely applicable screen for small molecules. This is useful in metabolic engineering, which is a promising approach to synthesizing compounds of pharmaceutical, agricultural, and industrial importance using bioengineered strains. There, the development of high-yield strains is often a costly and time-consuming process. This problem can be addressed by generating and testing large mutant strain libraries. However, a current key bottleneck is the lack of high-throughput screens to detect the small molecule products. The FP assay is quantitative, sensitive, fast, and cheap. As a proof of principle, we established the FP assay to screen for FK506 (tacrolimus) produced by Streptomyces tsukubaensis, which was cultivated in 96-well plates. An ultraviolet mutagenized library of 160 colonies was screened to identify strains showing higher FK506 productivities. The FP assay has the potential to be generalized to detect a wide range of other small molecules.

Super-multiplex vibrational imaging.

The ability to visualize directly a large number of distinct molecular species inside cells is increasingly essential for understanding complex systems and processes. Even though existing methods have successfully been used to explore structure-function relationships in nervous systems, to profile RNA in situ, to reveal the heterogeneity of tumour microenvironments and to study dynamic macromolecular assembly, it remains challenging to image many species with high selectivity and sensitivity under biological conditions. For instance, fluorescence microscopy faces a 'colour barrier', owing to the intrinsically broad (about 1,500 inverse centimetres) and featureless nature of fluorescence spectra that limits the number of resolvable colours to two to five (or seven to nine if using complicated instrumentation and analysis). Spontaneous Raman microscopy probes vibrational transitions with much narrower resonances (peak width of about 10 inverse centimetres) and so does not suffer from this problem, but weak signals make many bio-imaging applications impossible. Although surface-enhanced Raman scattering offers high sensitivity and multiplicity, it cannot be readily used to image specific molecular targets quantitatively inside live cells. Here we use stimulated Raman scattering under electronic pre-resonance conditions to image target molecules inside living cells with very high vibrational selectivity and sensitivity (down to 250 nanomolar with a time constant of 1 millisecond). We create a palette of triple-bond-conjugated near-infrared dyes that each displays a single peak in the cell-silent Raman spectral window; when combined with available fluorescent probes, this palette provides 24 resolvable colours, with the potential for further expansion. Proof-of-principle experiments on neuronal co-cultures and brain tissues reveal cell-type-dependent heterogeneities in DNA and protein metabolism under physiological and pathological conditions, underscoring the potential of this 24-colour (super-multiplex) optical imaging approach for elucidating intricate interactions in complex biological systems.

Synthesis of photoactivatable azido-acyl caged oxazine fluorophores for live-cell imaging.

We report the design and synthesis of a photoactivatable azido-acyl oxazine fluorophore. Photoactivation is achieved cleanly and rapidly with UV light, producing a single fluorescent oxazine photoproduct. We demonstrate the utility of azido-acyl caged oxazines for protein specific labeling in living mammalian cells using the TMP-tag technology.

Cooperative Vinculin Binding to Talin Mapped by Time-Resolved Super Resolution Microscopy.

The dimeric focal adhesion protein talin contains up to 22 cryptic vinculin binding sites that are exposed by unfolding. Using a novel method to monitor the in situ dynamics of the talin dimer stretch, we find that in contrast to several prevalent talin dimer models the integrin-binding talin N-termini are separated by 162 ± 44 nm on average whereas as expected the C-terminal dimerization domains colocalize and are mobile. Using vinculin tagged by DHFR-TMP Atto655 label, we found that optimal vinculin and vinculin head binding occurred when talin was stretched to 180 nm, while the controls did not bind to talin. Surprisingly, multiple vinculins bound within a single second in narrowly localized regions of the talin rod during stretching. We suggest that talin stretches as an antiparallel dimer and that activates vinculin binding in a cooperative manner, consistent with the stabilization of folded talin by other binding proteins.