Plants have long served as natural indicators of environmental conditions, and recent advances in synthetic biology are enabling the design of engineered sentinels - living sensors that can report on abiotic and biotic stressors. This review summarizes recent advances in designing sensor plants, also called phytosensors or sentinel plants, highlighting three major strategies: (1) exploiting native promoter systems responsive to environmental cues, (2) engineering protein-based genetically encoded biosensors that detect specific molecules of interest, and (3) constructing interkingdom signaling networks between plants and microbes to extend sensing capabilities to the rhizosphere. These sense-response modules can be coupled to optical reporters (e.g., fluorescence, bioluminescence, and pigment-based) that enable remote detection via drones and satellite imaging. Continued improvements in promoter design, receptor modularity, and signal visualization technologies are driving the development of robust, field-deployable plant biosensors. Together, these innovations position engineered sensor plants as scalable, self-sustaining sentinels for real-time environmental monitoring and land management.

Agave, which thrives in arid regions of North Americas, is a promising alternative feedstock for biofuel production due to its high sugar content and drought tolerance. A major challenge to using agave as a feedstock for bioprocessing is its toxicity. Saponins, natively produced glycosides, disrupt cell membranes and are highly toxic to Saccharomyces cerevisiae even at low concentrations. To enable agave as a feedstock, we combined rational engineering with adaptive laboratory evolution (ALE) to create new S. cerevisiae strains with high tolerance to agave extracts. Heterologous overexpression of tomatinase from Fusarium oxysporum improved tolerance up to 10% agave extracts. In parallel, 27 serial passages of ALE resulted in evolved strains that can grow in 4% extracts and produce ethanol equivalent to the starting strains. Whole genome sequencing revealed mutations in coding sequences of multidrug efflux pumps (PDR5 and PDR1) and a non-coding sequence adjacent to α-glucosidase gene (IMA3), suggesting roles in resistance to saponins. Combining both approaches — tomatinase overexpression and evolved strains — yielded a high-tolerant strain that produced ethanol in 20% agave extracts. Our dual approach to strain engineering demonstrates an effective multi-level strategy of developing microbial hosts with increased resistances to toxicity feedstock components, ultimately enabling new bioprocesses.

The ExFAB biofoundry seeks to develop automated workflows to discover, phenotype, and prototype biology from extreme and exceptional fungi, archaea, and bacteria. Central to many of ExFAB's workflows is robotic liquid handling. This protocol describes the start-up procedure for ExFAB's Tecan Fluent 780 system, equipment that underpins automated workflows ranging from liquid sample preparations to microbial culturing. Covered in this protocol is the start-up procedure for the Fluent system as well as the associated peripheral equipment. Together this system includes: Tecan Fluent 780 Work Station with 8 channel pipetting head, 96 channel head, heating, cooling, shaking, and magnetic blocks; Tecan Spark UV/Vis Plate Reader; Rotanta 460 Robotic Centrifuge; and, Liconic StoreX STX44 Incubator. This protocol can be used in conjunction with other protocols dedicated to automated liquid handling workflows including ExFAB workflows for automated phenotyping, prototyping, and discovery.

Nitazenes are an emergent class of synthetic opioids that often rival or exceed fentanyl in their potency. These compounds have been detected internationally in illicit drugs and are the cause of increasing numbers of hospitalizations and overdoses. New analogs are consistently released, making detection challenging — new ways of testing a wide range of nitazenes and their metabolic products are urgently needed. Here, we develop a computational protocol to redesign the plant abscisic acid receptor PYR1 to bind diverse nitazenes and maintain its dynamic transduction mechanism. The best design has a low nanomolar limit of detection in vitro against nitazene and menitazene. Deep mutational scanning yielded sensors able to recognize a range of clinically relevant nitazenes and the common metabolic byproduct in a complex biological matrix with limited cross-specificity against unrelated opioids. Application of protein design tools on privileged receptors like PYR1 may yield general sensors for a wide range of applications in vitro and in vivo. Nitazenes are potent synthetic opioids that are difficult to detect. Here, authors computationally redesign a plant receptor to create sensitive sensors capable of detecting diverse nitazenes and their metabolites in biological samples.

Abstract The design of maximally divergent DNA sequences translating into the same protein is a critical problem in synthetic biology. Current design tools that rely on heuristics or machine learning often fail to effectively minimize the length of shared subsequences between the gene copies, compromising strain stability. Here, we introduce SIRIUS, a combinatorial optimization algorithm designed to generate maximally divergent coding sequences for a given protein of interest. Leveraging integer linear programming enforcing host-specific codon usage thresholds, SIRIUS stabilizes synthetic constructs and broadens the accessible design space for robust and scalable synethtic biology. Experimental results show that SIRIUS produces diverse sequences with fewer shared subsequences than existing methods. SIRIUS is freely available on GitHub at https://github.com/ucrbioinfo/sirius .

Small-molecule sensing in plants is dominated by chemical-induced dimerization modules. In the abscisic acid (ABA) system, allosteric receptors recruit phosphatase effectors and achieve nM in vivo responses from µM receptor-ligand interactions. This sensitivity amplification could enable ABA receptors to serve as generic scaffolds for designing small-molecule sensors. To test this, we screened collections of mutant ABA-receptors against 2,726 drugs and other ligands and identified 553 sensors for 6.6% of these ligands. The mutational patterns indicate strong selection for ligand-specific binding pockets. We used these data to develop a sensor design pipeline and isolated sensors for multiple plant natural products, 2,4,6-trinitrotoluene (TNT), and "forever" per- and polyfluoroalkyl substances (PFAS). Thus, the ABA sensor system enables design and isolation of small-molecule sensors with broad chemical scope and antibody-like simplicity.

Methods for fast and inexpensive gene synthesis from oligonucleotide pools enable rapid iteration of genetic designs. Here, we describe iggypop (indexed Golden Gate gene assembly from PCR-amplified oligonucleotide pools), a simple computational-experimental pipeline that allows for low-cost design and synthesis of hundreds of genes from oligonucleotide pools using Golden Gate assembly methods. We used iggypop to synthesize a series of single-transcript autonomously bioluminescent reporters (STARBURSTs) that link the five genes of a fungal bioluminescence pathway via ribosomal skipping LP4/2A sequences into a 9.5 kb transcript that function in planta. We also synthesized RUBY reporters (a reporter gene system producing red betalain pigment) recoded to match dicot codon usage, as RUBY was codon optimized for rice codon usage and has a high GC content. Surprisingly, the recoded RUBYs substantially reduced betalain production in transient Nicotiana benthamiana assays. Based on this observation, we synthesized six GC-boosted STARBURSTs, which produced robust luminescence in both transient assays and transgenic Arabidopsis plants. Thus, iggypop enabled the rapid synthesis of multiple genetic designs to deliver a bright single transcript autobioluminescent reporter. Iggypop should enable the facile synthesis and optimization of new genetic parts and complex polycistronic pathways.

Designing CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) single guide RNA (sgRNA) libraries targeting entire kingdoms of life will significantly advance genetic research in diverse and underexplored taxa. Current sgRNA design tools are often species-specific and fail to scale to large, phylogenetically diverse datasets, limiting their applicability to comparative genomics, evolutionary studies, and biotechnology. Here, we introduce ALLEGRO, a combinatorial optimization algorithm designed to compose minimal, yet highly effective sgRNA libraries targeting thousands of species at the same time. Leveraging integer linear programming, ALLEGRO identified compact sgRNA sets simultaneously targeting multiple genes of interest for over 2000 species across the fungal kingdom. We experimentally validated sgRNAs designed by ALLEGRO in Kluyveromyces marxianus, Komagataella phaffii, Yarrowia lipolytica, and Saccharomyces cerevisiae, confirming successful genome edits. Additionally, we employed a generalized Cas9-ribonucleoprotein delivery system to apply ALLEGRO's sgRNA libraries to untested fungal genomes, such as Rhodotorula araucariae. Our experimental findings, together with cross-validation, demonstrate that ALLEGRO facilitates efficient CRISPR genome editing, enabling the development of universal sgRNA libraries applicable to entire taxonomic groups.

The production of high value and commodity chemicals, biopharmaceuticals and biofuels using Saccharomyces cerevisiae is hindered by various stress factors that affect yield and efficiency. Tardigrades, known for their remarkable stress tolerance, express unique proteins responsible for their resilience. This study evaluates the impact of expressing the tardigrade proteins CAHS3, MAHS, and RvLEAM on stress tolerance in S. cerevisiae. Our results show that high yields of these proteins do not impede yeast growth, except for CAHS3, which reduces proliferation. Expression of MAHS enhances acute heat tolerance, while MAHS and RvLEAM confer increased tolerance to acute hyperosmotic stress. Both CAHS3 and RvLEAM improve desiccation survival. However, these proteins do not provide benefits under chronic stress conditions such as prolonged exposure to high temperature, hyperosmotic stress, or solvents. These findings highlight the potential utility of tardigrade proteins for transient stress protection in industrial bioprocesses and suggest future engineering approaches for improved stress tolerance in yeast.

Designing CRISPR single guide RNA (sgRNA) libraries targeting entire kingdoms of life will significantly advance genetic research in diverse and underexplored taxa. Current sgRNA design tools are often species-specific and fail to scale to large, phylogenetically diverse datasets, limiting their applicability to comparative genomics, evolutionary studies, and biotechnology. Here, we present ALLEGRO, a combinatorial optimization algorithm able to design minimal, yet highly effective sgRNA libraries targeting thousands of species. Leveraging integer linear programming, ALLEGRO identified compact sgRNA sets simultaneously targeting several genes of interest for over 2,000 species across the fungal kingdom. We experimentally validated the sgRNAs designed by ALLEGRO in Kluyveromyces marxianus , Komagataella phaffii , and Yarrowia lipolytica . In addition, we adopted a generalized Cas9-Ribonucleoprotein delivery system coupled with protoplast transformation to extend ALLEGRO's sgRNA libraries to other untested fungal genomes, such as Rhodotorula araucariae . Our experimental results, along with cross-validation, show that ALLEGRO enables efficient CRISPR genome editing, supporting the development of universal sgRNA libraries applicable to entire taxonomic groups. ### Competing Interest Statement The authors have declared no competing interest.

Rational control over diverse, ligand-responsive output networks is a foundational challenge in synthetic biology, particularly for systems based on post-translational signaling. Here, we present the design and engineering of minimal, modular protein architectures for chemically responsive molecular inverters and digital switches. Our inverter design modifies the plant-derived PYR1-HAB1 chemically inducible dimerization module by incorporating a constitutive activator, which is then competitively displaced by the ligand-bound PYR1 receptor, converting the native ‘dimerization-on’ mechanism into a ‘signal-off’ inverter. We establish key design features and demonstrate predictable tuning of the inverter’s transfer function, including its maximum output, half-maximal inhibitory concentration, and minimum output, solely by adjusting protein stoichiometry. The architecture is modular, enabling plug-and-play response to diverse, user-defined drug-like small molecules. We show that an inverter biosensor for an environmental contaminant functions in engineered living cells with a low nanomolar sensitivity. Additionally, we convert the PYR1-HAB1 sensor into a digital switch by adding an engineered molecular titrant to the system. Overall, this work provides a generalizable, minimal, and tunable protein scaffold for programming complex, post-translational signaling logic, significantly expanding the toolkit for sophisticated biological circuit design.

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas genome-wide screens are powerful tools for unraveling genotype-phenotype relationships, enabling precise manipulation of genes to study and engineer industrially useful traits. Traditional genetic methods, such as random mutagenesis or RNA interference, often lack the specificity and scalability required for large-scale functional genomic screens. CRISPR systems overcome these limitations by offering precision gene targeting and manipulation, allowing for high-throughput investigations into gene function and interactions. Recent work has shown that CRISPR genome editing is widely adaptable to several yeast species, many of which have natural traits suited for industrial biotechnology. In this review, we discuss recent advances in yeast functional genomics, emphasizing advancements made with CRISPR tools. We discuss how the development and optimization of CRISPR genome-wide screens have enabled a host-first approach to metabolic engineering, which takes advantage of the natural traits of nonconventional yeast-fast growth rates, high stress tolerance, and novel metabolism-to create new production hosts. Lastly, we discuss future directions, including automation and biosensor-driven screens, to enhance high-throughput CRISPR-enabled yeast engineering.

The production of recombinant proteins requires the precise coordination of various biological processes, including protein synthesis, folding, trafficking, and secretion. The overproduction of a heterologous protein can impose various bottlenecks on these networks. Identifying and alleviating these bottlenecks can guide strain engineering efforts to enhance protein production. The methylotrophic yeast Komagataella phaffii is used for its high capacity to produce recombinant proteins. Here, we use ribosome profiling to identify bottlenecks in protein secretion during heterologous expression of human serum albumin (HSA). Validation of this analysis showed that the knockout of non-essential genes whose gene products target the ER, through co- and post-translational mechanisms, and have high ribosome utilization can increase production of a heterologous protein, HSA. A triple knockout in co-translationally translocated carbohydrate and acetate transporter Gal2p, cell wall maintenance protein Ydr134cp, and the post-translationally translocated cell wall protein Aoa65896.1 increased HSA production by 35 %. This data-driven strain engineering approach uses cell-level information to identify gene targets for phenotype improvement. This specific case identifies hits and creates strains with improved HSA production, with Ribo-seq and bioinformatic analysis to identify non-essential ER targeted proteins that are high ribosome utilizers.

Rhodotorula , a red yeast genus inhabiting diverse environmental and clinical niches, is a promising microbial source for carotenoid and lipid production. Despite the coexistence of sexual and asexual species, the evolutionary mechanisms underlying their reproductive diversity remain poorly understood. Here, we present a global genomic survey of 249 Rhodotorula strains using Oxford Nanopore, PacBio, and Illumina sequencing. Phylogenomic analysis resolved a robust species tree, delineating three major clades with substantial cryptic species diversity. Chromosome-level assemblies revealed a constrained tetrapolar mating system characterized by unlinked pheromone/receptor ( P/R ) and homeodomain ( HD ) mating-type ( MAT ) loci but displaying infrequent recombination. This genomic architecture is widely conserved, even in species lacking observed sexual cycles, suggesting widespread sexual potential. The P/R locus functions as a supergene, exhibiting conserved gene content and order within mating types but variable configurations between compatible types. Clade-specific biases in mating-type allele distributions indicates asymmetric evolutionary pressures. Structural rearrangements and trans-specific polymorphism of mating-specific allele at the P/R locus, may drive sequence divergence, potentially generating novel mating compatibility within the same mating type. The intermediate P/R genomic states retaining pheromone genes from the opposite mating type may preserve residual functions or facilitate transitions in mating-type architecture. Together with the presence of homozygous hybrid strains, these genomic signatures suggest a potential shift toward same-sex-like mating. Our findings elucidate how mating-type architecture and allele dynamics underpin the evolution of reproductive strategies in fungi and suggest that innovations in MAT system may be a key evolutionary lever in fungal adaptation across environments. Significance Statement Rhodotorula yeasts are a widespread genus and occupy a unique intersection of biology serving both as emerging fungal pathogens and industrial workhorses producing carotenoids and lipids. Yet, despite their growing importance, the genetic mechanisms shaping their reproductive strategies remain elusive. By leveraging a global dataset of 249 genomes, we uncover the hidden architecture of sexual identity in Rhodotorula : a conserved but flexible MAT locus structure, varying degrees of recombination suppression, and lineage-specific mating-type imbalances. Our discovery of same-sex-like mating signatures and widespread interspecies hybrids reveals a dynamic evolutionary landscape, where sexual plasticity fuels adaptation, speciation, and possibly pathogenesis. These insights redefine our understanding of sexual evolution in basidiomycetous yeasts and open new avenues for biotechnology and medical mycology. ### Competing Interest Statement The authors have declared no competing interest. Chinese Academy of Sciences, CAS-TAX-24-021, CAS Scholarship U.S. National Science Foundation, IOS-2134912, EF-2125066, DBI-2400327, DBI-1429826, DBI-2215705 National Institutes of Health, R01 AI130128, S10-OD016290 National Institute of Food and Agriculture, 2020-70029-33202 Canadian Institute for Advanced Research Fellow, Jason E. Stajich The Slovenian Research and Innovation Agency, MRIC UL, I0-0022, P4-0432, P1-0198, J4-60078

Monoterpenes are valued for their roles as flavors, fragrances, insecticides, and energy-dense fuels. Microorganisms provide sustainable biosynthesis routes for these important molecules, but production levels remain limited. Here, we introduce a biosensor-driven microbial engineering strategy to enhance monoterpene production, specifically targeting geraniol. Using mutagenized libraries of the PYR1 receptor─a versatile biosensor from plant ABA signaling pathways with a malleable binding pocket─we screened 24 monoterpenes and identified PYR1 variants responsive to eight, including geraniol. A low background, highly selective geraniol-sensitive PYR1 variant was expressed in the thermotolerant yeast Kluyveromyces marxianus as a growth-based biosensor circuit, allowing for rapid strain engineering. By coupling the geraniol-sensitive PYR1 sensor with a genome-wide CRISPR-Cas9 mutagenesis approach, we identified six gene knockouts that enhance geraniol production, achieving up to a 2-fold increase in titer. This study demonstrates the power of the PYR1 biosensor platform to enable rapid strain engineering and the identification of mutants that improve the titer of a desired metabolite.

Plants sense abscisic acid (ABA) using chemical-induced dimerization (CID) modules, including the receptor PYR1 and HAB1, a phosphatase inhibited by ligand-activated PYR1. This system is unique because of the relative ease with which ligand recognition can be reprogrammed. To expand the PYR1 system, we designed an orthogonal '*' module, which harbors a dimer interface salt bridge; X-ray crystallographic, biochemical and in vivo analyses confirm its orthogonality. We used this module to create PYR1*MANDI/HAB1* and PYR1*AZIN/HAB1*, which possess nanomolar sensitivities to their activating ligands mandipropamid and azinphos-ethyl. Experiments in Arabidopsis thaliana and Saccharomyces cerevisiae demonstrate the sensitive detection of banned organophosphate contaminants using living biosensors and the construction of multi-input/output genetic circuits. Our new modules enable ligand-programmable multi-channel CID systems for plant and eukaryotic synthetic biology that can empower new plant-based and microbe-based sensing modalities.

Precise control of gene expression is critical for optimizing cellular metabolism and improving the production of valuable biochemicals. However, hard-wired approaches to pathway engineering, such as optimizing promoters, can take time and effort. Moreover, limited tools exist for controlling gene regulation in non-conventional hosts. Here, we develop a two-channel chemically-regulated gene expression system for the multi-stress tolerant yeast Kluyveromyces marxianus and use it to tune ethyl acetate production, a native metabolite produced at high titers in this yeast. To achieve this, we repurposed the plant hormone sensing modules (PYR1ABA/HAB1 and PYR1*MANDI/HAB1*) for high dynamic-range gene activation and repression controlled by either abscisic acid (ABA) or mandipropamid (mandi). To redirect metabolic flux towards ethyl acetate biosynthesis, we simultaneously repress pyruvate dehydrogenase (PDA1) and activate pyruvate decarboxylase (PDC1) to enhance ethyl acetate titers. Thus, we have developed new tools for chemically tuning gene expression in K. marxianus and S. cerevisiae that should be deployable across many non-conventional eukaryotic hosts.

Class II Type V endonucleases have increasingly been adapted to develop sophisticated and easily accessible synthetic biology tools for genome editing, transcriptional regulation, and functional genomic screening in a wide range of organisms. One such endonuclease, Cas12a, presents itself as an attractive alternative to Cas9-based systems. The ability to mature its own guide RNAs (gRNAs) from a single transcript has been leveraged for easy multiplexing, and its lack of requirement of a tracrRNA element, also allows for short gRNA expression cassettes. To extend these functionalities into the industrially relevant oleaginous yeast Yarrowia lipolytica, we developed a set of CRISPR-Cas12a vectors for easy multiplexed gene knockout, repression, and activation. We further extended the utility of this CRISPR-Cas12a system to functional genomic screening by constructing a genome-wide guide library targeting every gene with an eightfold coverage. Pooled CRISPR screens conducted with this library were used to profile Cas12a guide activities and develop a machine learning algorithm that could accurately predict highly efficient Cas12a gRNA. In this protocols chapter, we first present a method by which protein coding genes may be functionally disrupted via indel formation with CRISPR-Cas12a systems. Further, we describe how Cas12a fused to a transcriptional regulator can be used in conjunction with shortened gRNA to achieve transcriptional repression or activation. Finally, we describe the design, cloning, and validation of a genome-wide library as well as a protocol for the execution of a pooled CRISPR screen, to determine guide activity profiles in a genome-wide context in Y. lipolytica. The tools and strategies discussed here expand the list of available synthetic biology tools for facile genome engineering in this industrially important host.

CRISPR-Cas9 functional genomic screens uncover gene targets linked to various phenotypes for metabolic engineering with remarkable efficiency. However, these genome-wide screens face a number of design challenges, including variable guide RNA activity, ensuring sufficient genome coverage, and maintaining high transformation efficiencies to ensure full library representation. These challenges are prevalent in non-conventional yeast, many of which exhibit traits that are well suited to metabolic engineering and bioprocessing. To address these hurdles in the oleaginous yeast Yarrowia lipolytica, , we designed a compact, high-activity genomewide sgRNA library. The library was designed using DeepGuide, a sgRNA activity prediction algorithm and a large dataset of similar to 50,000 sgRNAs with known activity. Three guides per gene enables redundant targeting of 98.8% of genes in the genome in a library of 23,900 sgRNAs. We deployed the optimized library to uncover genes essential to the tolerance of acetate, a promising alternative carbon source, and various hydrocarbons present in many waste streams. Our screens yielded several gene knockouts that improve acetate tolerance on their own and as double knockouts in media containing acetate as the sole carbon source. Analysis of the hydrocarbon screens revealed genes related to fatty acid and alkane metabolism in Y. lipolytica. The optimized CRISPR gRNA library and its successful use in Y. lipolytica led to the discovery of alternative carbon source-related genes and provides a workflow for creating high-activity, compact genome-wide libraries for strain engineering.