Degenerative genome evolution is widely found among obligatory bacterial mutualists, as observed in plant-sucking hemipteran insects whose symbiont genomes are highly reduced and specialized for provisioning of essential amino acids. Originally, such symbionts must have been derived from environmental free-living bacteria. It is elusive, however, what evolutionary changes are involved in the early stages of such elaborate mutualistic associations. Here, we addressed this evolutionary question using the experimental symbiotic system consisting of the stinkbug Plautia stali and the model bacterium E. coli. In E. coli, metJ encodes a repressor of the methionine synthesis pathway, and its disruption upregulates production of the essential amino acid methionine. We found that, when metJ-disrupted E. coli was inoculated to P. stali, the insects exhibited significantly elevated hemolymphal methionine levels and improved adult emergence rates, demonstrating that the single-gene mutation makes E. coli mutualistic to P. stali. In comparison with mutualistic E. coli single-gene mutants that upregulate another essential amino acid tryptophan, the phenotypic effects on P. stali were somewhat different: the adult emergence rate was improved by both the methionine-overproducing and tryptophan-overproducing E. coli mutants, whereas the adult body color was improved by the tryptophan-overproducing E. coli mutant only. When we generated a double mutant E. coli ΔmetJΔtnaA and inoculated it to P. stali, the adult emergence rate was not improved but rather attenuated, uncovering non-additive fitness consequences of these single-gene mutations. These results provide insights into what genetic changes may have facilitated the early evolution of the insect-microbe mutualism.

Saccharomyces cerevisiae is widely adopted as a chassis in synthetic biology. However, heterologous constructs often disrupt proteostasis, metabolism, redox balance, and secretory processes. These disruptions activate a complex network of stress pathways. These include the heat shock response, unfolded protein response, oxidative stress defenses, cell wall integrity signaling, the high-osmolarity glycerol pathway, and Snf1/AMPK-mediated energy regulation. Collectively, these pathways form a stressome that maintains cellular homeostasis but constrains productive capacity. A comprehensive understanding of how synthetic designs interact with these pathways is essential for developing robust yeast systems. Strategies such as promoter tuning, chaperone augmentation, redox and cofactor balancing, lipid and membrane optimization, dynamic regulation, and pathway compartmentalization can reduce cellular burden. Emerging methods also improve stress mitigation. These include CRISPR-based circuit rewiring, adaptive laboratory evolution, synthetic organelle construction, and data-driven strain engineering. This review summarizes construct-induced stress in engineered yeast and presents stress-aware design principles to advance more resilient, higher-yielding S. cerevisiae strains for biotechnology.

Plant roots host defined microbial communities that differ from those found in the surrounding soil and these communities shift dynamically in response to plant development and environmental changes. Whilst it is widely accepted that root exudates play a key role in the assembly and dynamics of root-associated microbial communities, the underlying mechanisms are not well understood. This is partly due to a lack of controlled experimental systems that monitor both exudate- and microbiome-dynamics simultaneously. Here, we compared two microcosm systems commonly used in either root microbiome (clay particle-based) or root exudate studies (glass bead-based) for their suitability to simultaneously monitor both aspects. We evaluated these systems based on plant performance, bacterial growth, and time-resolved community and exudate profiling. In both systems, we reveal an exudate effect, characterized by higher bacterial diversity and Pseudomonas abundances in proximity to plant roots. While clay particles impeded exudate recovery, even when plants were removed from microcosms for exudate collection, the glass bead set-up allowed us to uncover dynamic exudate shifts during bacterial community establishment. This highlighted a transient increase of glucosinolates upon root colonization by initially dominant Pseudomonas species. Overall, the comparison proved only the glass bead-based semi-hydroponic system to be suitable for the paralleled study of exudate and root microbiome dynamics.

Translational regulation offers a powerful biological control axis with the potential to enable programmable control over synthetic mRNAs. Here, we introduce inducible Deaminases Acting on RNA (iDARs): deaminase domains (DDs) with conditional RNA-editing activities. Using a domain-insertion strategy, we designed autoinhibited enzymes that can be converted into active RNA editors in response to triggers based on small molecules (chemiDARs), intracellular antigens (antiDARs), protease cleavage (lysiDAR), and optical excitation (optiDAR). Coupling these domains with novel stop codon containing RNA substrates enabled conditional protein translation or transcript degradation. Mutational tuning of inositol hexaphosphate (IP6)-binding pockets produced tightly regulated deaminases with minimal basal activity, facilitating dose-dependent readthrough translation in response to low-nanomolar drug concentration, with dynamic ranges exceeding 100-fold. By encoding iDARs alongside their substrates, we developed 'self-editing' polycistronic transcripts capable of directing translation of encoded proteins in a trigger-dependent manner following delivery to cells as in vitro transcribed mRNAs. Overall, iDARs provide a generalizable framework for generating controllable deaminases, enabling the design of post-transcriptional circuits that link biochemical sensing to readouts based on de novo translation or mRNA decay.

Selective encapsulation of target enzymes is an increasingly well studied field with a host of potential applications for biotechnology. Natively, many bacteria utilize bacterial microcompartments (BMC) for enzyme encapsulation to enhance catalysis. BMCs are protein shells that enable selective localization of targeted metabolic enzymes and may improve catalytic rates by co-localizing pathway enzymes and/or serve to sequester toxic or volatile intermediates. The microcompartment shell of Haliangium ochraceum (HO) is a notable BMC chassis because of its modularity and versatility; it is easily expressed and assembled outside its native host and can accept a wide array of cargo. Recently, it was demonstrated that assembly of HO BMC shells can be easily achieved in vitro. Following up on our previous work on in vivo assembly of HO-BMCs with triose phosphate isomerase (TPI) as model enzyme cargo, here we have demonstrated the advantages of in vitro assembly (IVA) for targeted enzyme encapsulation. We achieved variable loading of BMC shells with targeted amounts of TPI and demonstrated enhanced thermal stability of encapsulated TPI versus free TPI up to 62°C.

Indole-3-acetic acid (IAA) is a tryptophan-derived gut microbial metabolite with reported anti-inflammatory activities, but the organisms and anaerobic pathways that support robust production remain unclear. Screening 206 human gut bacterial isolates by LC-MS revealed that IAA production is rare: only five strains exceeded the limit of quantitation, and high-capacity production was confined to the acetogens Blautia hydrogenotrophica and Intestinibacter bartlettii. Across growth conditions, IAA was a minor product that rose alongside carbohydrate-sensitive, OFOR-linked catabolism of multiple amino acids, generating abundant branched-chain and aromatic organic acids. In gnotobiotic mice mono-colonized with I. bartlettii, these metabolites were produced in vivo but showed distinct host handling, with branched-chain fatty acids largely extracted between portal and peripheral plasma, whereas aromatic acids and their glycine conjugates appeared in plasma and urine. Genomic analyses and heterologous enzyme assays identified expanded repertoires of 2-oxoacid:ferredoxin oxidoreductases (OFORs) with activities spanning pyruvate/oxaloacetate, branched-chain, and aromatic 2-oxoacids, including indolepyruvate conversion to indoleacetyl-CoA, a putative intermediate en route to IAA. Finally, position-specific 13C tracing showed that CO2 released during amino acid oxidation is reassimilated into acetate via reductive acetogenesis, indicating that gut acetogens can maintain redox balance without fermenting partner strains. Together, these findings show that high IAA output is restricted to select gut acetogens and linked to a broader OFOR-driven anaerobic metabolism that generates additional metabolites that are absorbed by the host.

Artificial biomolecular condensates have emerged as powerful tools to control cellular behaviors. Here we introduce a method to build artificial condensates within living mammalian cells through the design of modular RNA motifs formed by a single, short strand of RNA. These condensates emerge spontaneously, creating RNA-rich compartments that remain separated from their surrounding environment. The RNA sequences include stem-loop domains that fold as the RNA is transcribed, and then condense in the nucleus and cytoplasm through loop-loop interactions. These sequences can be optimized and diversified, enabling the generation of distinct, non-mixing condensate populations and the programmable control of their subcellular localization. The RNA motifs can also be modified to recruit small molecules, proteins, and RNA molecules in a sequence-specific manner to the RNA-rich phase. By introducing additional RNAs that link two distinct types of condensates, we can create droplets with multiple subcompartments, whose organization can be controlled by tuning the stoichiometry of different RNA sequences. These artificial condensates provide a versatile platform for studying and manipulating molecular functions inside living cells.

The nonconventional yeast, Yarrowia lipolytica, is a promising protein expression host, having achieved recombinant protein expression yield on par with the commonly used host, Komagatella phaffii (Pichia pastoris). However, strong, fully constitutive genetic elements and expression cassettes for protein expression in Y. lipolytica remain limited. In this study, we leveraged genome-wide transcriptomics to uncover five strong promoters and four terminators. Among these, the promoter of ribosomal protein L41 demonstrated superior activity to the strongest previously reported promoters. We further demonstrated the functionality of pL41 across different media conditions and by using it to express diverse heterologous proteins. Similarly, we showed that the terminator of glutathione-S-transferase (tGST) supported higher protein expression and low transcriptional readthrough compared to commonly used terminators. To support protein secretion efforts, we utilized a secretomics-guided signal peptide screen to unveil three signal peptides, demonstrating broad applicability to different proteins. Integrating these genetic elements into a new expression cassette (YALI-pSTOmics1) resulted in a 3-fold increase in secretory expression of bovine fibroblast growth factor 2 compared to a combination of the best available state-of-the-art genetic tools for gene expression in Y. lipolytica. This expression cassette represents an open-source alternative to expensive commercial ones. Furthermore, the novel promoters and terminators provide options for metabolic engineering, where reuse of existing genetic parts is often a limitation.

We present a microfluidic workflow that couples reconstituted in vitro transcription-translation (IVTT) with ultra-high throughput droplet screening to directly link genotype and phenotype within complex, heterogeneous DNA pools. The approach employs DNA nanoflowers as clonal, high-copy templates, enabling robust protein expression from single DNA molecules encapsulated in picoliter droplets. When integrated with fluorescence-assisted microdroplet sorting (FADS) and a DNA recovery pipeline that reconstituted selected libraries for subsequent iterative rounds, the platform achieves approximately 400-500 fold enrichment per selection cycle and supports functional discovery and directed evolution entirely independent of host cell expression. As a proof of principle, we demonstrate recovery of the recombinase RecA from an E. coli genomic library screened for single-stranded DNA binders, highlighting the platform's capability to identify DNA-interacting and DNA-modifying enzymes. By eliminating host-derived background activity and toxicity constraints that often complicate lysate or cell-based metagenomic screens, this method expands access to enzyme classes that have historically been difficult to assay.

Origin firing is a central process during DNA replication, but specific sequences defining replication origin usage have not been defined in human cells. Here, we show that a genome language model can accurately predict which sequences can act as an origin of replication, thereby enabling the fast and cost-effective creation of genome-wide replication origin maps. We fine-tuned a genome language model on the primary sequence of mapped human origins to establish ORILINX (ORIgin of replication Language-model Inference via Nucleotide conteXt) and found that it learns a rich representation of sequence features linked to replication initiation, extending beyond known predictive features such as GC-content and G-quadruplex motifs. When applied genome-wide, the model's sequence-derived origin calling closely mirrors origin efficiency inferred from replication timing, suggesting that intrinsic sequence context encodes information relevant to initiation frequency. Furthermore, we performed Short Nascent Strand sequencing (SNS-seq) and Repli-seq to demonstrate that ORILINX can generalize to other mammalian genomes, such as those of mice and sheep, as well as other vertebrates such as chickens. Finally, we packaged ORILINX into a simple, easy-to-use tool which is available at https://github.com/Pfuderer/ORILINX.git.

Probiotics provide multiple health benefits and are widely used in food, pharmaceutical, and agricultural applications. However, their viability and efficacy are severely compromised during production, storage, and application due to environmental stresses, including heat, dehydration, oxidation, acids, and bile salts, with mortality rates reaching up to 90% during industrial processing. Probiotic stress tolerance is a complex, multilayered physiological process involving cell membrane integrity, protein stability, stress-responsive signaling pathways, nucleic acid protection, osmoprotectant accumulation, and metabolic reprogramming. This review systematically summarizes the molecular mechanisms underlying probiotic stress tolerance and critically evaluates current enhancement strategies, including strain screening, adaptive evolution, genetic engineering, and encapsulation technologies. Furthermore, emerging approaches, such as CRISPR-based genome editing, synthetic biology, and nanomaterial-assisted delivery, are highlighted as promising solutions to overcome translational bottlenecks. At the same time, challenges related to regulatory approval and large-scale manufacturing remain to be addressed.

Volatile organic compounds (VOC) emitted by soil bacteria influence interactions with other soil microbes and with plant roots. While their potential as plant-growth promoters is well recognized, their role in promoting plant resilience to abiotic stress and the underlying molecular mechanisms remains poorly understood. Here, we investigate the role of Pseudomonas VOCs in enhancing plant resilience to drought stress. Arabidopsis thaliana plants were exposed to VOCs emitted by Pseudomonas strains under both control and osmotic-stress conditions. VOC exposure generally enhanced plant growth, and this effect was even more pronounced under both drought and salt stress. Transcriptomic analysis revealed that VOC exposure modulates key stress-responsive pathways, including those related to abscisic acid biosynthesis and signalling, sugar transport, iron uptake, aliphatic glucosinolate biosynthesis, and plant defences. Using Arabidopsis mutants, we identified abscisic acid and aliphatic glucosinolates as important components in mediating the plant response to VOCs. SWEET11/12 sugar transporters and ABA signaling genes were downregulated by VOCs exposure, in order to allow for a positive regulation of lateral root numbers (in case of SWEET genes) and plant growth in general under drought stress. In summary, using metabolomics, transcriptomics and functional analysis, we showed a negative cross-talk between the effects of VOCs on plant growth and glucosinolate production, whereas a positive interaction was observed between the biosynthesis of coumarins and VOCs. Notably, VOCs also improved drought tolerance in soil-grown Brassica oleracea plants. We showed that VOC treatment altered the root-associated microbiome under drought, leading to a community composition more similar to that of well-watered plants. Our results show that Pseudomonas emitted VOCs can promote plant growth under drought conditions, linked to root transcriptional reprogramming and direct or indirect microbiome modulation.

Agriculture is under pressure to provide food for a growing population and the feedstock required to drive the bioeconomy. Methods to breed and genetically modify plants are inadequate to keep pace. When engineering crops, traits are painstakingly introduced into plants one-at-a-time, combine unpredictably, and are continuously expressed. Synthetic biology is changing these paradigms with new genome construction tools, computer aided design (CAD), and artificial intelligence (AI). “Smart plants” contain circuits that respond to environmental change, alter morphology, or respond to threats. Further, the plant and associated microbes (fungi, bacteria, archaea) are now being viewed by genetic engineers as a holistic system. Historically, plant health has been enhanced by many natural and laboratory-evolved soil microbes marketed to enhance growth or provide nutrients, or pest/stress resistance. Synthetic biology has expanded the number of species that can be engineered, increased the complexity of engineered functions, controlled environmental release, and can assemble stable consortia. New CAD tools will manage genetic engineering projects spanning multiple plant genomes (nucleus, chloroplast, mitochondrion) and the thousands of genomes of associated bacteria/fungi. This review covers advanced genetic engineering techniques to drive the next agricultural revolution, as well as push plant engineering into new realms for manufacturing, infrastructure, sensing, and remediation.

Fold switching, where a protein region interconverts between entirely distinct three-dimensional structures, is emerging as vital for certain protein functions. Here, we report a remarkable example in the F7 pyocin, a phage tail-like bactericidal nanomachine. Cryogenic electron microscopy and tomography reveal that a 163-residue segment of the central tail fiber undergoes a dramatic transition—from a trimeric α-helical coiled-coil to a triangular β-prism—upon binding to the bacterial cell surface. This massive fold switch remodels the tail tip, ejects the internal tape measure protein, and drives membrane puncture. Site-directed mutations that selectively destabilize the β-prism conformation completely abolish bactericidal activity without impairing particle assembly, implying that the energy released during this transition powers penetration. AlphaFold-based analyses further predict similar large-scale coiled-coil to β-prism switches in diverse non-contractile phage tails. This discovery reveals a sophisticated, ATP-independent strategy for microbial warfare and opens exciting possibilities for engineering next-generation bacteriocins to combat multidrug-resistant pathogens.