Surfactin, a cyclic lipopeptide produced by Bacillus species, represents a promising bio-pesticide due to its antimicrobial activity, environmental compatibility, and ability to induce plant immunity. However, fragmented knowledge on its molecular mechanisms and engineering limits the rational development of next-generation biopesticides. This review outlines surfactin biosynthetic principles and advances in metabolic and synthetic engineering strategies for improving yield and structural diversity. We emphasize high-throughput NRPS reprogramming strategies enabling precise control over peptide sequence and fatty acid composition, providing a powerful platform for the rational design of functionally tailored variants. Furthermore, surfactin may serve as an important signaling hub in the interaction network between plants and microorganisms. By integrating NRPS engineering, system metabolic design, and the construction of synthetic microbial communities, the future of crop protection is expected to shift from a single active molecule application model to a programmable ecological regulation model.

Scalable methods for producing and characterizing protein libraries are essential for generating standardized datasets needed to train models linking sequence to function. Here, we present Amplicon/Protein Bead Display (APB-Display), which expresses and purifies >100,000 protein variants in vitro in 1 day. APB-Display uses particle-templated emulsification to generate libraries of hydrogel beads that covalently display many copies of a given protein variant and its encoding DNA. By incubating beads with fluorescently-labeled ligands, sorting, and sequencing to generate titration curves (APB-TiteSeq), we simultaneously quantified expression levels and binding affinities (Kds) for >18,000 FLAG epitope variants interacting with M2 anti-FLAG antibody in 3 days, revealing chemistry-dependent epistasis between positions 4 and 5. Single-concentration binding measurements (APB-SortSeq) paired with neural network denoising further returned quantitative affinities for >88,000 variants. APB-Display requires only standard laboratory equipment and access to a FACS sorter, providing an accessible platform for quantitative in vitro biochemistry at scale.

Genomovar-level and intragenomic diversity cannot be resolved by conventional amplicon sequencing due to the limitation of fragment lengths and read accuracy, while the application of metagenomic profiling to a large number of samples can be resource intensive. Here, we report UltraRes-rrn, an integrated wet-lab and computational workflow for high-accuracy rrn (i.e., 16S-ITS-23S rRNA) operon profiling using Nanopore sequencing. By integrating ultra-long DNA recovery, long-read amplicon sequencing, and unique molecular identifiers (UMI)-based consensus correction, UltraRes-rrn obtains full-length 16S-ITS-23S rRNA operon consensus sequences with mean accuracies exceeding 99.98%. To achieve higher resolution, we propose a hierarchical rRNA operon profiling strategy in which concatenated 16S+23S rRNA genes (16S23S) serve as a primary and the internal transcribed spacer (ITS) provides a secondary marker. The 16S23S marker achieved discrimination at the genomovar level compared to either 16S or 23S rRNA genes alone, which mitigates the ITS-driven over-splitting observed with the full-length rrn operon and allows for larger proportion of data being classified at higher confidence thresholds. Further, ITS variation was strongly structured by tRNA occurrence patterns, suggesting that ITS can capture taxon microdiversity missed by either 16S or 23S rRNA gene sequences alone. The UltraRes-rrn workflow was applied to full-scale nitrogen removal reactors, revealing intraspecies diversity variation driven by different carbon regimes, which would not have been possible with a shorter gene sequence. In summary, UltraRes-rrn enables cost-effective community profiling at genomovar-level resolution in complex ecosystems.

Central to CRISPR technologies is the single-guide RNA (sgRNA), an engineered fusion of a processed CRISPR RNA and tracrRNA that directs Cas9 and many Cas12 nucleases to bind and cleave target DNA. Here, we report the discovery of similarly compact viral sgRNAs (vsgRNAs) encoded by bacteriophages that counteract bacterial CRISPR-Cas9 immunity. vsgRNAs inhibit Cas9 function via two complementary routes: by sequestering the Cas9 apoenzyme, and by re-directing the Cas9 nuclease to transcriptionally silence its own promoter. vsgRNAs also can cooperate with co-encoded anti-CRISPR proteins (Acrs), including AcrIIA25.1 that blocks DNA binding by Cas9 complexed with a standard sgRNA but not with the vsgRNA. We predict that phages evolved vsgRNAs by co-opting and repurposing host-encoded long-form tracrRNAs (tracr-L) responsible for Cas9 auto-repression and a countermeasure to Acrs. Our search also uncovered Cas9-regulating small CRISPR-associated RNAs (scaRNAs), which we predict were inserted upstream of tracrRNAs to form tracr-L but were also co-opted by phages as viral scaRNAs to suppress Cas9 immunity. Finally, we found that vsgRNAs can enable genome editing in mammalian cells, offering a natural guide RNA template for CRISPR technologies. Overall, these findings reveal that bacteriophages devised their own compact sgRNAs tailored to subvert Cas9 immunity, long preceding their rational design to program RNA-guided nucleases.

The ever-expanding catalogue of uncharacterized proteins - the so called functional dark matter - poses a major challenge for biotechnological and biomedical exploitation. Functional assessment of most proteins is hindered by the technical limitations of annotation transfer and by the propagation of erroneous annotations in databases. The common denominator here is the reliance on sequence similarities. However, these become inaccurate below certain thresholds and can diverge even at sequence identities around 70%. To approach this challenge, we implemented a strategy using embeddings generated by protein language models for targeted function discovery (PE-TFD). Datasets of proteins representing target as well as non-target functions were used to train supervised learning models. The resulting ensemble models yielded interpretable prediction scores, enabling the exploration of databases without relying on multiple sequence alignments or structural information. We here tested PE-TFD for the discovery of novel hydrogenases as proof-of-concept, resulting in the novel discovery of 773 [NiFe] and 1,929 [FeFe] hydrogenases that were not detected by established sequence- or profile-based approaches. Structural analyses supported their non-random nature and further revealed a significant number of enzymes lacking prior functional annotation. Our framework therefore enables interpretable function discovery in large-scale datasets and the exploitation of functional dark matter.

5-Aminolevulinic acid (5-ALA) is a naturally occurring nonproteinogenic amino acid with considerable potential in agricultural and pharmaceutical applications, and its biomanufacturing has attracted growing interest. To meet market demand, this study developed an efficient production process by engineering E. coli. First, we established the C4 biosynthetic pathway for 5-ALA and minimized glycine degradation by targeted deletion of gcvP and kbL. Subsequently, the introduction of a nonoxidative glycolysis (NOG) module combined with enhanced glucose uptake further increased 5-ALA production. To alleviate host toxicity, efflux engineering and antioxidant systems were integrated, further raising the titer to 6.93 ± 0.28 g/L. Dynamic regulation of sucCD expression together with redox rebalancing maintained a balance between precursor supply and cell growth. Finally, attenuation of hemB expression reduced metabolic flux diversion toward heme synthesis, yielding the engineered strain A48, which produced 55.11 g/L of 5-ALA within 41 h in a 5 L bioreactor. The metabolic engineering strategy presented in this study establishes an efficient biosynthetic platform for 5-ALA production.

Heparin has been the most important drug for treating thrombotic disorders for more than 60 years. However, the traditional production of heparin involves the slaughter of animals. Therefore, there is a demand for the animal-free production of heparin, such as enzymatic synthesis based on the heparin biosynthetic pathway. To achieve this, robust 6-O-sulfotransferases (6OSTs) are required to produce the 6-O-sulfation pattern in heparin, which is crucial for the biological activity. However, most native 6OSTs are derived from animal tissues and exhibit poor recombinant expression, low catalytic efficiency, and insufficient stability in E. coli. To overcome these limitations, we systematically established a two-tier engineering framework that integrated structure-guided protein repair and optimization of translation. Cross-species screening identified Oryzias melastigma 6OST-1 as an engineering-competent template. First, we constructed the variant 6OST-M10 through protein repair one-stop service-guided structural restoration combined with targeted reverse mutations. To address the long-standing challenge of low heterologous expression, we generated a synonymous rare-codon (SRC)-guided ultrahigh-throughput screening platform based on split-GFP complementation. This platform systematically tunes the translation kinetics for sulfotransferases. Ultimately, the 6OST-M10(SRC) variant was generated, which displayed an 8.375-fold increase in soluble expression and a 27-fold improvement in catalytic activity that reached 4400 IU/L under high-density fermentation. This dual-layer strategy couples structural stabilization with translational optimization to resolve the trade-offs among activity, stability, and recombinant expression that have previously limited bacterial production of animal-derived sulfotransferases and heparin synthesis in E. coli. This framework provides a universal, scalable paradigm for protein engineering and enables the synthesis of bioengineered heparin and other glycosaminoglycan products without the involvement of animals.

Plasmids benefit bacterial communities by storing auxiliary genes that address environmental challenges such as antibiotics. Subsequent plasmid loss can also be advantageous if plasmid benefits are temporary but costs are permanent. However, unless positive selection is sustained, plasmid loss can proceed to extinction, with access to plasmid-derived benefits permanently lost. In principle, horizontal transmission can maintain a plasmid in a population, but if the plasmid cost is too high, the host can become uncompetitive. We examine how survival of costly but occasionally beneficial plasmids is possible in a bacterial population. Using population models, we demonstrate that plasmid-dependent phages can, counterintuitively, solve this plasmid survival problem for their bacterial hosts. Phage predation pins the plasmid at low but nonzero abundance, such that the plasmid cost is effectively neutralized at the population level, dramatically lengthening the persistence time of the plasmid. When conditions change and the costly plasmid becomes beneficial, it spreads across the host population and switches to a vertical-transmission lifestyle until benefits again subside.