Retrons and several defence-associated reverse transcriptases (DRTs) synthesize non-genomic DNA for bacteriophage immunity. In some instances, this non-genomic DNA is of undefined, semirandom sequence. How undefined DNA sequences impart antiphage defence is not known. Here we report the cryo-EM structure and functional characterization of the DRT1 antiphage defence system. We show that DRT1 performs template-free, protein-primed DNA synthesis to generate semirandom DNA adducts. DNA synthesis activates the nitrilase domain of DRT1 while DNA adducts drive assembly of quiescent DRT1 filaments. Filamentous DRT1 is comprised of domain-swapped C-termini that are entwined, forming pseudoknots between tetrameric stacks. This configuration occludes the apo-nitrilase active site, resulting in a dormant state. Bacteriophage escape mutants identify a T4 single-stranded DNA helicase required for DRT1 activity. Functionally, DRT1 resembles a minimal retron where a single gene produces an RT, effector, and non-genomic antitoxin DNA. 

Dormancy, characterized by an extended lag time before resuming growth, protects bacteria from antibiotic stress and facilitates the emergence of resistance. However, a common mechanism that precisely regulates bacterial lag time remains poorly defined. Here we show that the formation and disassembly dynamics of protein aggregates function as a proteostasis clock that governs the lag time distribution. By tracking the aggregate and regrowth dynamics of thousands of cells, we observed that dormant bacteria from diverse genetic and environmental contexts converge to delayed aggregate disassembly as a common rate-limiting step for growth resumption. Mechanistically, the chromosomal replication initiator DnaA is sequestered in aggregates and spatially separated from the nucleoid, thereby preventing DNA replication until proteostasis restoration enables aggregate disassembly. We modulated bacterial lag time distribution by genetically and chemically targeting the proteostasis-replication axis. Remarkably, lag extension scales linearly with the disruption level of proteostasis across diverse backgrounds, indicating a universal coupling between dormancy and proteostasis. This proteostasis collapse renders dormant bacteria, including clinical isolates, more sensitive to additional proteotoxic stress. These findings reveal a timekeeping mechanism in cellular dormancy, highlighting proteostasis as a shared target for combating antibiotic tolerance.

Microbial competition for trace metals shapes their communities and interactions with humans and plants. Many bacteria scavenge trace metals with metallophores, small molecules that chelate environmental metal ions. Metallophore production may be predicted by genome mining, where genomes are scanned for homologs of known biosynthetic gene clusters (BGC). However, accurately detecting non-ribosomal peptide (NRP) metallophore biosynthesis requires expert manual inspection, stymieing large-scale investigations. Here, we introduce automated identification of NRP metallophore BGCs through a comprehensive algorithm, implemented in antiSMASH, that detects chelator biosynthesis genes with 97% precision and 78% recall against manual curation. We showcase the utility of the detection algorithm by experimentally characterizing metallophores from several taxa. High-throughput NRP metallophore BGC detection enabled metallophore detection across 69,929 genomes spanning the bacterial kingdom. We predict that 25% of all bacterial non-ribosomal peptide synthetases NRPS encode metallophore production and that significant chemical diversity remains undiscovered. A reconstructed evolutionary history of NRP metallophores supports that some chelating groups may predate the Great Oxygenation Event. The inclusion of NRP metallophore detection in antiSMASH will aid non-expert researchers and continue to facilitate large-scale investigations into metallophore biology.

Adipic acid (AA) is an important dicarboxylic acid that serves as a precursor in the synthesis of nylon-6,6. Given the increasing market demand for AA and the environmental concerns associated with its conventional production, the development of sustainable biosynthetic techniques for AA production has become a key research focus in both industry and academia. However, industrially viable technologies for AA biosynthesis remain constrained by several challenges, particularly incomplete raw material utilization, low strain conversion efficiency, a complex fermentation process, and high costs of downstream separation. To overcome these barriers, this review presents the current state of AA biosynthesis, critically discussing biosynthetic pathways and advanced metabolic engineering strategies and tools for constructing cell factories with high conversion efficiency. The basic principles relevant to improving the fermentation process and downstream separation technologies are also comprehensively reviewed. Key challenges and knowledge gaps are identified, providing insights to guide future research toward commercially viable biobased AA production.

Insulin glargine, a long-acting insulin analog, is essential for diabetes treatment. However, its industrial production remains challenging due to limitations in conventional expression systems. Here, we employed the industrial filamentous fungus Trichoderma reesei as an alternative expression host for insulin glargine production, leveraging its superior protein secretion capacity. Initially, expression constructs containing the constitutive Pcdna1 promoter and CBH1 signal peptide (SP1) showed successful transcription but failed to achieve extracellular secretion, presumably due to induced endoplasmic reticulum (ER) stress. Consequently, we implemented a fusion protein strategy utilizing three distinct carrier proteins (CBH1, CBH2, and LA-20) to enhance glargine secretion. Notably, the CBH1 fusion not only enabled detectable glargine secretion but also significantly alleviated the ER stress. Furthermore, replacement of SP1 with the Aspergillus niger β-glucoamylase (glaA) signal peptide achieved a fourfold enhancement in glargine secretion and further reduced cellular stress responses. Following these systematic optimizations, a final yield of 58.95 mIU/L glargine was achieved in shake-flask cultures. Thus, the combined strategy described here could achieve extracellular production of glargine in T. reesei, suggesting that it is a promising host for secretory production of therapeutic recombinant proteins, particularly complex analogs like glargine.

The analysis of ribosome profiling (Ribo-Seq) data has provided evidence that many eukaryotic mRNAs contain translated upstream or downstream ORFs (uORFs/dORFs), but the biological significance of this translation activity remains, for the most part, unknown. One of the principal limitations has been the lack of Ribo-Seq data from several closely related species, precluding the identification of cases in which translation is phylogenetically conserved. Here, by combining Ribo-Seq data from 100 different experiments, we identify 2,332 translated uORFs and 1,008 translated dORFs in S. cerevisiae, which result in microproteins that tend to be highly hydrophobic or positively charged. To study their phylogenetic conservation, we have generated Nanopore direct RNA sequencing data, together with Ribo-Seq data, from six additional Saccharomyces species, spanning an evolutionary period of around 16 million years. We have identified 195 translated S. cerevisiae uORFs that are also translated in other Saccharomyces species; these uORFs are translated at levels comparable to the main coding sequence and display signatures of purifying selection at the level of the encoded microproteins. In contrast, dORFs are translated at very low levels and they are rarely conserved, suggesting much more limited microprotein functionalization. We have also discovered that uORF translation is associated with the formation of alternative transcript isoforms encompassing the region containing the uORFs but not the main protein coding sequence, implying that some microproteins can be produced independently of the main protein product. This work significantly advances our understanding of how initially pervasive uORF translation can result in new microproteins, providing many new candidates for further functional studies.