Plasmids are the workhorses of molecular biology: fast, flexible, and often taken for granted. We clone, overexpress, tag, and mutate freely, assuming they will faithfully produce RNA transcripts that match the intended DNA sequence. This assumption is rarely tested and often invalidated. Sequences in plasmid backbones, epitope tags, and codon-optimized regions may inadvertently harbor cryptic promoters or splice sites. The resulting unexpected transcripts and proteins, while often undetected, can distort results and propagate false conclusions through papers, grants, and even clinical trials. In this perspective, we highlight published cases where plasmids have distorted results and misled interpretation. We examine the mechanisms and consequences of plasmid-associated expression artifacts and offer practical strategies to minimize them. Finally, we call for a revision of community standards for experiments using transgenes: deposit complete plasmid sequences and verify the resulting transcripts using RNA-seq.

Bacteriocins are ribosomally synthesized antimicrobial peptides produced by various classes of bacteria, exhibiting broad-spectrum activity that makes them promising candidates for applications in food preservation and medicine. Their inherent stability under extreme pH, temperature, and salinity conditions further supports their functional versatility. However, the widespread industrial application of bacteriocins remains constrained by the low titres typically achieved during fermentation. Despite extensive efforts to optimize production using batch and fed-batch fermentation strategies, the resulting titres remain inadequate for economically viable large-scale manufacturing. This review aims to provide a comprehensive overview of novel process strategies developed over the past two decades to enhance bacteriocin yields during fermentation. One of the primary challenges is the inhibition of microbial growth due to the accumulation of lactic acid or the bacteriocin itself during production. To address this, in-situ product removal techniques—such as co-cultivation with lactic acid-consuming microorganisms, in-situ adsorption, filtration, and foam fractionation—have been explored, yielding notable improvements in bacteriocin titres. Additionally, stress-induced production strategies involving biological (e.g. co-culture with competing microbes), chemical (e.g. salinity and pH stress), and physical (e.g. agitation, temperature, and aeration stress) stimuli have also demonstrated success in enhancing bacteriocin synthesis.  This review underscores the importance of these innovative fermentation approaches and highlights the need for further research focused on scaling up such processes. Advancing these strategies is critical to realizing the full potential of bacteriocins in food safety, antimicrobial therapy, and broader biomedical applications.

When environmental bacteria transition to laboratory conditions, a process termed domestication, the shift from the native habitat to a culture medium often reduces cell cultivability. Consequently, most bacteria remain uncultured using standard techniques, leaving the majority of their diversity unexplored. Here we introduce an enhanced domestication (EDEN) method for bacterial cultivation, which acclimatises environmental bacteria to culture media through a controlled and gradual exposure. To facilitate EDEN, we develop a 3D-printable microwell plate incorporating growth chambers integrated with a continuous-flow media reservoir. Using amplicon sequencing, we show that EDEN-acclimatised bacterial polycultures grow as distinct populations with significantly greater diversity and likely-uncultured taxa compared with standard cultivation methods. Similarly, EDEN-acclimatised bacterial monocultures show threefold greater diversity and tenfold more likely-uncultured taxa. EDEN also doubled the cultivability of agarose-encapsulated microcolonies. Finally, we demonstrate the utility of EDEN by isolating a previously uncultured bacterium exhibiting broad-spectrum antimicrobial activity against drug-resistant pathogens.

The commercialization of cultivated meat remains limited by the high cost of cell culture medium. Here, we discuss how multi-omics characterization and computational modelling can enable the design of cell line-specific, serum-free medium using valorized sources, offering improved cost-efficiency and sustainability.

Type III CRISPR–Cas systems confer antiviral immunity via cyclic oligoadenylate (cOA) signaling. Here, we elucidate a cooperative bacterial defense strategy involving two cOA-activated CRISPR-associated Rossmann fold (CARF)-containing effectors, adenosine deaminase CAAD and ribonuclease Csx1, in Thermoanaerobaculum aquaticum. Genomic analyses indicate widespread co-occurrence of CRISPR-associated adenosine deaminase (CAAD) with ancillary CARF-containing effectors in type III CRISPR systems, suggesting that multiple CARF-containing proteins may contribute to a coordinated cOA-dependent defense. Biochemical and structural studies reveal the intrinsic dynamics of CAAD hexamer, and demonstrate that cA4/cA6 binding stabilizes CAAD hexamers, triggering metal-ion-dependent conversion of ATP into inosine triphosphate. Concurrently, the downstream Csx1 is exclusively activated by cA4 to cleave single-stranded RNA. Strikingly, we found that both effectors are capable of degrading cA4, suggesting that this CAAD–Csx1 pair may be cross-regulated and achieve immunity through a dual-targeting mechanism: in response to infection, Csx1 degrades viral RNA while CAAD disrupts nucleotide metabolism via ATP deamination, which can be relieved via cA4 degradation when infection has been eliminated. This study proposes an enhanced defense mechanism through coordinated activation and regulation of multiple CRISPR effectors by a single signaling molecule, unveiling unprecedented complexity in CRISPR immunoregulation.

The social behaviors of microbes provide unique opportunities for testing social evolution theories. How can altruistic behaviors arise by natural selection is a central challenge in biology. Green-beard effect has been proposed as a basic mechanism for the evolution of altruistic behaviors. Yet, green-beard genes are generally thought to be rare. Here, we find that the Schizosaccharomyces pombe gsf2 gene mediates flocculation-like aggregation, and flocculation is triggered by acid stresses. gsf2-expressing cells preferentially adhere to each other. The expression of gsf2 is costly, but gsf2-expressing cells preferentially adhere to each other and protect each other from external stress. Gsf2 is highly variable in natural populations, likely contributing to different flocculation intensity. These findings suggest that gsf2 is a gradient green-beard gene that drives the altruism among gsf2 carriers. Moreover, we find that gsf2 is a new gene that originated very recently. Our results provide insights into the origin and evolution of green-beard genes.

Pyomelanin has been extensively applied in various fields. However, the yield of pyomelanin isolated from natural producers is low. Engineering microbial biosynthesis is considered a sustainable and economically feasible alternative. We utilize engineered Komagataella phaffii to increase the yield of homogentisic acid by 66-fold by balancing three different biosynthetic modules and further synthesizing pyomelanin by oxidative polymerization. During this process, we establish a pyomelanin color screening system and apply this system to perform directed evolution of 3-deoxy-D-arabinoheptulosonate 7-phosphate synthase and semirational design modification of hydroxyphenylpyruvate dioxygenase. We report the difference of pyomelanin between oxidative polymerization under alkaline conditions and that under laccase treatment. After high-density fermentation in a 5-L fermenter for 204 h, strain Pyo29 achieve the highest titer (70.5 ± 0.7 g/L). Finally, we demonstrate the potential application value of pyomelanin. This study provides a feasible solution for high-yield pyomelanin biosynthesis with substantial industrial production. Pyomelanin is resistant to UV light, and has antibacterial and anti-inflammatory effects, however, the yield of pyomelanin isolated from natural producers is low. Here the authors engineer Komagataella phaffii to produce 70.5 g/L and isolate the product.

Lignin forms the polyphenolic network in wood that enables trees to stand upright, transport water and ions, and resist microbial attack. Although its structural complexity is often seen as a limitation, its inherent multifunctionality offers opportunities. By strategically harnessing these features, new directions for advanced lignin-based materials can emerge.