Reverse genetic approaches in molds are complicated by their recalcitrance to transformation, multicellular growth during much of the vegetative life cycle, and frequent off-target integration of deletion constructs. Thus, protocols for gene deletion in filamentous fungi are predominantly confined to established model organisms. Adapting an existing protocol developed for gene deletion in the related fungus Aspergillus fumigatus, we employed an expression-free CRISPR/Cas9 directed mutagenesis strategy to the non-model environmental mold Aspergillus calidoustus. As a test case, we have deleted A. calidoustus pyrG, encoding orotidine-5'-phosphate decarboxylase, using short regions of homology to guide on-target integration of a nourseothricin resistance cassette (NatR) to CRISPR/Cas9-induced double strand breaks. We genotypically and phenotypically validated two A. calidoustus ΔpyrG deletion strains generated using this methodology, with whole-genome sequencing revealing one ΔpyrG strain to have integrated the resistance cassette by homologous recombination, and another strain by non-homologous end joining. Thus, distinct modes of double-strand break repair were responsible for on-target integration of the homology-bearing NatR cassette at the pyrG locus in A. calidoustus.IMPORTANCEIn the environment, filamentous fungi play essential roles in soil health and agriculture, decomposition, and nutrient cycling. The study of these organisms is often limited by an inability to dissect the functions of their genes and the roles that they play by modifying the genomes of these organisms. Here, we adapt and validate a tool for genome modification to Aspergillus calidoustus, a soil fungus that forms a partnership with a bacterium (Paraburkholderia edwinii) to resist natural toxins found in the soil.

The respiratory Complex I is a highly intricate redox-driven proton pump that powers oxidative phosphorylation across all domains of life. Yet, despite major efforts, its long-range energy transduction principles remain much debated. Here, we study the molecular principles of proton transport by engineering the antiporter modules of Complex I. By combining directed mutagenesis with time-resolved spectroscopy and molecular dynamics (MD) simulations, we identify conserved residues along the proton channels that control the rate of proton transfer across proteoliposome membranes. The antiporter modules catalyze this tightly regulated proton transport by transient water wires that follow intrinsic electric fields along the proton channels. Based on MD simulations, we identify conserved gating sites, established by nonpolar residues, which modulate the hydration and electric field effects underlying the proton transport upon mutation. On a general level, our findings highlight how the modular energy-transduction machinery of Complex I employs a combination of electrostatic and conformational coupling principles to catalyze long-range proton transport, with distinct similarities to other enzymes.

Ribonucleotide reductases (RNRs) catalyze the conversion of ribonucleotide (RNA) to deoxyribonucleotide (DNA) building blocks initiated by a long-range (>30 Å) proton-coupled electron transfer (PCET) by mechanistic principles that remain much debated. By combining multiscale quantum and classical simulations with directed mutagenesis, X-ray crystallography, and vibrational and electron paramagnetic resonance spectroscopy, we elucidate here the molecular principles underlying how metal-free RNRs initiate the long-range PCET process by creating a highly stable 3,4-dihydroxyphenylalanine (DOPA) initiator radical. We show that DOPA• is redox-tuned by a low-barrier hydrogen bond (LBHB), with a delocalized proton that provides the catalytic power for the ribonucleotide reduction. We find that the LBHB couples to an extended hydrogen-bonded network, with distant mutations resulting in the loss of radical formation, and providing key molecular insight into the long-range radical transport mechanism in RNRs. On a general level, our findings support the direct involvement of LBHB in protein chemistry and the importance of quantum effects in enzyme catalysis.

Sterols are a class of lipids that play a crucial role in human health through their essential physiological roles and as a point of interaction between commensal and pathogenic bacteria. The biosynthesis and modification of these lipids is a well-characterized process in many eukaryotes and increasingly in bacteria. However, the proteins responsible for formation of the unusual 8(14)-unsaturation found in the sterols produced by aerobic methanotrophs, dinoflagellates, nematodes, and marine sponges, remains unknown. Here, we utilize a heterologous expression system to identify a bacterial 8,14-sterol reductase (8,14-Bsr) responsible for generating the 8(14)-unsaturation in the aerobic methanotroph Methylococcus capsulatus. This enzyme modifies the direct product of C-14 demethylation, reducing one double bond in the nuclear core structure and isomerizing the other to produce an 8(14)-sterol. We subsequently tested the requirement of putative active site residues for catalysis through site directed mutagenesis, identifying residues likely involved in interacting with the sterol substrate and directly catalyzing this reaction. Bioinformatic analysis of the distribution of 8,14-Bsr reveals it is unique to the bacterial domain, found primarily in the Methylococcaceae family, the Mycobacteriales order, and yet uncultured members of the Myxococcota phylum. Further phylogenetic analysis of 8,14-Bsr suggests it shares an evolutionary history with the C-14 demethylase in these organisms and that these two enzymes were likely inherited together. These results provide insight into novel sterol biochemistry, further delimiting sterol biosynthesis in the bacterial domain from eukaryotes and illustrating the importance of molecular characterization to identify bacterial proteins that interact with sterols.

Pseudomonas putida JM37 as a novel bacterial chassis for ethylene glycol upcycling.

The projected surge in the global population, concomitant with escalating environmental perturbations, necessitates the sustainable production of high quality food to ensure long term food security. In order to overcome this major undertaking, adaptation of innovative, resilient and science driven agricultural strategies has been considered to be an essential step in plant biotechnology. Advanced biotechnological approaches like genome editing with site directed mutagenesis permit to step forward towards new cultivar with desirable traits in economically important crops Latterly, Zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspersed short palindromic repeats(CRISPR)/CRISPR associated protein 9 system has revolutionised the plant breeding system with exceptional achievements. On that account, the present review re-evaluates and discusses the information on genome editing technologies specially CRISPR/Cas9 approach in detailtogether with complete mechanism of CRISPR/Cas, problem and future aspects of the approach aiming to develop desirable agronomic genetic traits.

Genetically encoded green calcium indicators (GECIs) are broadly used for visualizing calcium transients in living cells. Among the diverse family of green GECIs, the Troponin C-based family offers potential advantages, including reduced calcium buffering, smaller molecular size, linear calcium response, and low cytotoxicity. However, the Troponin C-based GECIs with inverted calcium response are less developed compared to other popular GECIs, including GCaMPs and GECOs, and, as a consequence, have several drawbacks related to low dynamic range, brightness, photostability, and calcium ion sensitivity. To address these limitations, we developed a novel GECI, called icBTnC2, which incorporates Troponin C as a sensing moiety and the new bright photostable green FP mBaoJin as a reporting moiety. icBTnC2 demonstrated an inverted fluorescent response to calcium ion binding with a Kd of 62 nM. In terms of fluorescence contrast and calcium ion affinity in vitro, icBTnC2 was comparable to the best widely used calmodulin-based GECIs from the GCaMP family. icBTnC2 demonstrated superior photostability under wide-field fluorescence microscopy exhibiting 5.5-, 4.8-, 3.2-, 2.9-, and 1.3-fold higher photobleaching half-time compared to iYTnC2, mEGFP, NCaMP7, jGCaMP8f, and mBaoJin, respectively. The icBTnC2 indicator was benchmarked against other GECIs, such as jGCaMP8f, NCaMP7, iYTnC2, and R-GECO1, for visualization of calcium transients in mammalian cells and primary neuron cultures, and tested for calcium-dependent changes in fluorescence lifetime. Finally, we solved the crystal structure of the icBTnC2 indicator at 1.55 Å resolution in the calcium-bound state and, using directed mutagenesis, proposed the molecular basis of its fluorescent response to calcium ion binding.

ABSTRACT African Swine Fever virus (ASFV) causes a contagious and fatal disease in domestic pigs and Eurasian wild boars, representing a serious threat to the global pig industry, since no antivirals are available and vaccine use is currently restricted to Vietnam. Notably, ASFV encodes for an E2 ubiquitin-conjugating enzyme (ASFV-pI215L) which is essential for viral replication and evasion from immune interferon type I responses, suggesting that its functional impairment could lead to a live attenuated vaccine. In this study, we showed that ASFV-pI215L is highly conserved among 222 ASFV isolates, including the emerging ones, emphasizing its value as a target for vaccine design. Furthermore, our mutagenic studies revealed that single- and multiple-residue substitutions comprising the R11-E15 and D130-S134 residues reduced ASFV-pI215L E2 ubiquitin-conjugating activity. In parallel, a strong immunodominant B-cell epitope was mapped and mutated between P61 and F69 resides, reducing or abolishing both IgG and IgM recognition, and ASFV-pI215L E2 activity. In sum, this study highlights that rational targeted mutagenesis can reduce E2 ubiquitin-conjugating activity and immune recognition of ASFV-pI215L, providing a strategy to develop an attenuated vaccine able to differentiate infected from vaccinated animals.

In ovulo nucellus based direct somatic embryogenesis (DSE) is an efficient tool to assess mutagenic efficiency in perennial crops. A study was carried out in 2022–23 and 2023–24 at ICAR-Indian Agricultural Research Institute, New Delhi to compare the influence of 0.1% ethyl methane sulphonate (EMS) for 5 h and gamma irradiation (80 Gy) on embryogenesis, plantlet regeneration, and growth traits in kinnow mandarin (Citrus nobilis × Citrus deliciosa). Both treatments significantly reduced ovule survival compared with control (86.67%). The survival declined to 48.00% under EMS (0.1% for 5 h) and 35.78% following gamma irradiation (80 Gy for 10.9 min). The frequency of direct somatic embryogenesis declined to 49.07% under EMS and 43.85% under gamma irradiation. In contrast, post embryogenic parameters, including germination and bipolar conversion efficiency, were more severely affected, decreasing to 39.06–41.89% under EMS and 32.87–30.14%, under gamma treatment. Regenerants displayed short stature, reduced internodal length, and smaller leaves, though leaf number remained unaffected, suggesting that stress mainly altered expansion and elongation rather than leaf initiation. Correlation analysis indicated that biomass under EMS was closely linked to leaf area, while gamma treatment weakened relationships between height related traits and biomass. Cluster analysis revealed moderate variability under EMS but greater divergence among gamma irradiated populations. Overall, EMS generated point mutations with moderate phenotypic effects, whereas gamma rays exerted stronger disruptive impacts on regeneration and variability. These findings demonstrate that in ovulo nucellus-derived DSE is a robust system for evaluating mutagenic efficiency and optimizing treatments for generating targeted variability in perennial fruit crops.

A hydrolase with synergistic dual-cavity remodeling for high-efficiency degradation of PET waste.

Bile salts are conjugated steroids with digestive functions in vertebrates that reach the ecosystem upon excretion. Their environmental degradation by bacteria resembles the steroid nucleus catabolism that uses the 9,10-seco pathway, although there are two variants depending on whether the hydroxyl group at C-7 is eliminated (variant Δ4,6) or not (variant Δ1,4). Caenibius tardaugens, formerly known as Novosphingobium tardaugens, is a steroid-degrading bacterium used as a model to study the genetic and metabolic traits of steroidal sex-hormones catabolism. In this work, we investigated the bacterium ability to grow on bile salts such as cholate and deoxycholate and we performed directed mutagenesis along with transcriptomic analysis to shed light on the genes involved in bile salt metabolism. The mutation of the igr-like operon (EGO55_03105-EGO55_03125), similar to the cholesterol-degrading operon igr from Rhodococcus jostii RHA1, did not affect the ability to grow on bile salts. The transcriptomic analysis in the presence of cholate showed the induction of two gene clusters named bsd I (bile-salts degradation) (EGO55_16295 to EGO55_16335) and bsd II (EGO55_11460–EGO55_11480), containing genes that, according to their sequence identity to other bile salt-degrading bacteria, might participate in the side chain degradation and the HIP pathway of cholate catabolism, respectively. Moreover, the presence of other proteins homologous to the 7α-hydroxy steroid dehydratase Hsh2, such as EGO55_02245, EGO55_12965, or EGO55_06935, indicates that C. tardaugens cholate metabolism proceeds via the Δ4,6 variant, as it is conserved in several bacteria from the genera Sphingobium, Novosphingobium, and Sphingomonas.Supplementary InformationThe online version contains supplementary material available at 10.1007/s10532-026-10252-7.

Engineering T7 RNA polymerase-cascaded systems controlled by nisin and theophylline for protein overexpression and targeted gene mutagenesis in Lactococcus lactis.

Conjugal transfer of plasmids between bacteria is a major route for the spread of antimicrobial resistance. Many conjugative plasmids encode exclusion systems that inhibit redundant conjugation. In incompatibility group F (IncF) plasmids surface exclusion is mediated by the outer membrane protein TraT. Here we report the cryoEM structure of the TraT exclusion protein complex from the canonical F plasmid of Escherichia coli. TraT is a hollow homodecamer shaped like a chef’s hat. In contrast to most outer membrane proteins, TraT spans the outer membrane using transmembrane α-helices. We develop a microscopy-based conjugation assay to probe the effects of directed mutagenesis on TraT. Our analysis provides no support for the idea that TraT has specific interactions with partner proteins. Instead, we infer that TraT is most likely to function by physical interference with conjugation. This work provides structural insight into a natural inhibitor of microbial gene transfer.

Phosphorylation is an important protein post-translational modification and a valuable tool in medicinal chemistry for improving the pharmacological properties of small molecules, but rare in natural product biosynthesis. Here we report phosphorylation in cyanobactins, a family of ribosomally synthesized and post-translationally modified peptides. We identify an unusual kinase domain embedded in the C-terminal macrocyclase encoded in cyanobactin biosynthetic gene clusters and link it to the production of phosphorylated dolichospermamides and aphanizomenamides. Heterologous expression, domain deletion and site directed mutagenesis confirm the role of the kinase domain, while transplantation of this domain into the sphaerocyclamide pathway leads to the production of phosphorylated sphaerocyclamides. The kinase domain acts on both linear and cyclic substrates with preference for Tyr residues. Collectively, these findings expand the diversity of post-translational modifications in cyanobactins and establish a versatile strategy for the engineering and combinatorial biosynthesis of phosphorylated peptides.

Anthracyclines induce epigenetic changes in ALDH1A that promote chemoresistance in AML, revealing a potential treatment strategy co-targeting ALDH1A inhibition with hypomethylating agents

Chaperones ensure protein homeostasis and are conserved across species. The ATP-dependent chaperone Hsp90 is present from bacteria to eukaryotes, where it stabilizes and activates a wide range of substrate proteins called clients. However, what determines whether a protein depends on Hsp90 remains an open question. Here, we focused on the bacterial chaperone Hsp90 and its obligate client TilS (referred to as TilSSo) in the bacterium Shewanella oneidensis. Although Hsp90 is indispensable in S. oneidensis under heat stress by protecting the essential protein TilSSo from degradation by the protease HslUV, Hsp90 is dispensable in Escherichia coli, suggesting that E. coli TilS (TilSEc) is Hsp90 independent. We therefore compared the TilS orthologs with respect to in vitro stability, in vivo degradation, and interaction with Hsp90 to identify determinants of Hsp90 dependence. We found that in contrast to TilSSo, TilSEc was more stable, was not degraded by protease in the absence of Hsp90, and did not interact with Hsp90, indicating that TilSEc is not a client of Hsp90. Chimeras between TilSSo and TilSEc as well as directed mutagenesis revealed a region of TilSSo that is key for protease degradation and Hsp90 protection. Consistent with these results, the growth of S. oneidensis producing TilSEc was no longer dependent on Hsp90 under heat stress. Conversely, Hsp90 became essential for the growth of E. coli that produced TilSSo instead of TilSEc. Altogether, our work reveals that protein-specific features, such as stability and degradation sensitivity, can determine whether orthologous proteins require the bacterial Hsp90 chaperone in vivo.

HistENCODE: a proposed project to decipher functional interactions among and between histone PTMs.

With the imminent threat of nuclear arms race acceleration, and in the context of ongoing colonial nuclear violences, this paper seeks to trace the biological consequences for lives altered by the atom bomb, through Karen Barad’s agential realist ontology. To do this, I first present the paradigmatic development in the field researching the effects of radiation exposure, from direct genetic mutagenesis to the contribution of epigenetic responsivity. Based on the congruence of the epigenetic model with agential realism, I forward a speculative yet scientifically informed interpretation of epigenetic mechanisms as materially manifesting multiple temporalities of entangled events. To give a felt sense of the implications of this interpretation, it is diffractively read through a profound semi-autobiographic literary moment in Kyōko Hayashi’s novella From Trinity to Trinity, a testimony of the transformative effect of recontexualizing memories through their entanglements, as told by a survivor of the atomic bomb dropped on Nagasaki in 1945. By introducing a different conceptualization of epigenetic function, I aim to share my deep appreciation of the richness of cellular biology, and to stir new imaginings congruent with the relational, entangled nature of epigenetic mechanisms. Thought through the processing of trauma, it may offer possibilities of living and dying otherwise.

Deletion of FAM76B histidine-rich region enhances macrophage-mediated osteosarcoma inhibition via liquid-liquid phase separation.

RuBisCO (Ribulose-1,5-bisphosphate carboxylase/oxygenase) is the primary enzyme responsible for carbon fixation in the Calvin-Benson-Bassham cycle, yet it remains one of the most inefficient enzymes in nature. Its slow catalytic rate, low specificity for CO₂ over O₂, and susceptibility to inhibition and heat stress limit photosynthetic efficiency and crop productivity, particularly under fluctuating environmental conditions. This review highlights current strategies to enhance RuBisCO performance. These include direct mutagenesis, ancestral protein reconstruction, and the incorporation of chimeric or heterologous RuBisCO subunits to improve kinetics and stability. Enhancing the expression of native or foreign RuBisCO subunits, as well as engineering thermostable RuBisCO activase, further supports improved photosynthetic capacity. Additionally, the integration of carbon-concentrating mechanisms such as cyanobacterial carboxysomes and algal pyrenoids offers promising avenues to increase CO₂ availability around RuBisCO. Emerging synthetic biology approaches, including artificial carbon fixation pathways, aim to bypass the limitations of natural photosynthesis altogether. We have also discussed the limitations of each method. Complementing these advances, artificial intelligence and machine learning are being increasingly used to predict beneficial mutations, model enzyme behaviour, and guide protein engineering. Together, these multidisciplinary strategies hold great potential to optimize RuBisCO, improve crop yields, and enhance resilience in a changing climate.