Syngas conversion to carboxylic acids can be achieved through the combined actions of acetogens and chain elongators. Mixed cultures enriched with these functional groups can serve as robust and resilient catalysts for bio-based production, but developing efficient and stable communities remains challenging. In this study, we aimed to enrich a mixed microbial culture for efficient syngas-driven chain elongation using carbon monoxide (CO) and acetate as substrates. Three strategies were employed: (i) conventional enrichment using anaerobic sludge only, (ii) sludge augmentation with known syngas fermenters (Clostridium autoethanogenum, Clostridium carboxidivorans, Eubacterium limosum, and Acetobacterium wieringae) to enhance carboxydotrophic activity, and (iii) sludge augmentation with chain elongator Clostridium kluyveri to stimulate chain elongation. Enrichments were performed in batch cultures with stepwise increases in CO partial pressure across successive transfers until a 100% CO (170 kPa) headspace was reached. Ultimately, all enrichments grew under a 100% CO headspace, simultaneously utilizing CO and acetate, with butyrate as main product. 16S rRNA gene and metagenomic analyses revealed that Eubacterium became predominant. Several genera (Aminobacterium, Anaerotignum, Oscillibacter, and Proteiniphilum) not known to ferment CO, were also enriched. Based on physiological and metagenomic analyses, we hypothesize that Eubacterium consumes CO and acetate, producing butyrate and extracellular polymeric substance (EPS)-like material. Metagenomic data further suggests that components derived from this EPS-like material may serve as substrate for the other enriched genera. This study demonstrates that augmentation can steer mixed microbial communities toward targeted product formation and highlights the critical role of initial culture conditions in shaping community composition and functionality. 

CRISPR-based RNA cleavage holds promise for gene knockdown but its applications in metabolic engineering remain largely unexplored. Here we reconstitute three mammalian CRISPR-based RNA cleavage systems to arrest the intermediate metabolic reactions in the yeast Candida viswanathii to produce hydroxydecanoic acid (HDA), an important nylon precursor. We identify hfCas13d as the optimal effector and next develop a circular guide RNA (CgRNA) precursor architecture to enhance and extend the knockdown of genes involved in HDA production. This hfCas13d/CgRNA precursor platform also represses genes in other yeasts and E. coli, demonstrating broad microbial applicability. Single and dual CgRNA precursor arrays enable multiplexed pathway repression, with dual arrays reducing byproduct formation and increasing HDA titer to 17.8 g/L in shake flasks. Bioreactor fermentation further boosts HDA production to 37.2 g/L. Together, this work establishes hfCas13d/CgRNA as a programmable RNA regulatory platform for engineering microbial cell factories. CRISPR-based RNA cleavage holds promise for gene knockdown but its applications in metabolic engineering remain largely unexplored. Here the authors develop a circular guide RNA architecture to enhance the knockdown of genes involved in HDA production.

SauUSI is a dimeric, ATP-dependent type IV restriction enzyme that protects Staphylococcus aureus by cleaving nonself DNA containing 5-methylcytosine or 5-hydroxymethylcytosine. Using biophysical, single-molecule and nanopore sequencing methods, we show that 5-methylcytosine recognition first induces ATP-driven unidirectional translocation by one helicase-like subunit, displacing its target recognition domain (TRD). In half of events, the partner TRD can then bind the liberated modified site, stabilizing a growing DNA loop. On symmetrically methylated DNA, looping is more frequent and both motors can engage in bidirectional loop translocation. Cleavage occurs stochastically throughout translocation but events that increase dwell time such as rebinding methylated sites elevate the chance of cleavage. Interactions with other SauUSI dimers or protein roadblocks influence cleavage distributions but are not required for nuclease activation. While the first cleavages principally generate 1-nt 3ʹ overhangs or blunt ends, multiple binding–translocation–cleavage cycles by individual enzymes ultimately fragment the modified nonself DNA, neutralizing its threat. Using single-molecule physics and biochemical assays, Shaw, Hughes-Games and Torres Montaguth et al. reveal how the bacterial defense enzyme SauUSI repeatedly scans and makes multiple DNA breaks, efficiently fragmenting invading genetic material.

Structural variants (SVs) are a major source of genomic diversity, yet their discovery remains challenging due to repetitive genomic contexts, alignment ambiguity and the trade-off between sequencing cost and read length. Here we introduce HitSV, which implements repetitiveness- and signature-density-aware breakpoint recognition coupled with precise haplotype-resolved local assembly, thereby enabling base-resolution SV reconstruction and genotyping in various sequencing technologies. On the HG002 benchmark, HitSV achieved 15–72% (long read), 10–36% (short read) and 12% (hybrid sequencing), respectively, higher genotype F1 scores than state-of-the-art SV callers. Applied to the 1KGP phase 4 cohort, HitSV expanded the SV callset by 31.5%, substantially reshaping allele-frequency landscapes. In a large Chinese long-read cohort, HitSV uncovered tandem-repeat-mobile-element composite arrays as a prevalent, multi-allelic class of complex SVs, highlighting composite repeat architectures as an important and previously underrecognized contributor to human structural variation. HitSV models the interaction between genomic repetitiveness and signature intensity to improve structural variant detection.

Elucidating the intracellular organization of bacterial transcriptomes may reveal new modes of gene regulation. Recent work reported that chromosomal gene position determines mRNA localization within E. coli cells, implying a widespread genome-guided organizing mechanism. Yet this interpretation contrasts with biophysical expectations based on translation and diffusion and may be confounded by nascent transcription. Here, we examine this phenomenon by analyzing intracellular spatial transcriptomics datasets spanning different methods, bacterial species, growth conditions, and thousands of genes. We confirm the presence of genome-associated localization signatures that track chromosome macrodomain dynamics across the cell cycle and establish causality by repositioning a gene at distinct chromosomal loci. Quantitative analysis, however, shows that these signatures generally involve only a minority of transcripts and are of magnitudes consistent with reported rates of nascent transcription. Moreover, genome-associated patterns preferentially occur in weakly translated and unstable mRNAs. Together, our results indicate that genome-guided localization is unlikely to be a dominant driver of transcript positioning in bacteria. Genome organization has been previously proposed to dictate mRNA localization in bacteria. Here, the authors use spatial transcriptomic analysis to show that the genomic effect involves only a minority of transcripts, mainly weakly translated and unstable mRNAs.