The biological conversion of methanol into valuable chemicals represents a promising route for achieving global carbon neutrality. However, the rational engineering of complex industrial traits in microbial cell factories is hindered by an incomplete understanding of cellular metabolism and regulation. In this study, we developed a Random Mutagenesis Platform for Accelerated Genome Evolution (RAMPAGE) for the yeast Pichia pastoris (Komagataella phaffii). This platform enables continuous genome-wide diversification and facilitates the rapid evolution of superior phenotypes under defined selective pressures. Using RAMPAGE, we evolved yeast strains with significantly enhanced tolerance to extreme industrial conditions, including growth in 18% methanol, survival at 40 °C, and resistance to methanol and thermal stresses. Genome resequencing of these strains revealed a collection of genes associated with methanol tolerance and thermotolerance. In addition, we used RAMPAGE to evolve a glycoengineered yeast strain with improved growth and stress tolerance. Subsequent genome resequencing and functional validation identified novel genetic engineering targets to improve the cellular fitness of glycoengineered yeast strains. By providing a simple, efficient, and programmable tool for continuous genome evolution, RAMPAGE expands the synthetic biology toolkit for P. pastoris, enabling its broad application in industrial biotechnology.

3-Carene is a bicyclic monoterpene with a pine aroma. It is widely used as a flavoring agent, food additive, and insecticide in food and agricultural applications. Therefore, establishing an efficient microbial pathway for 3-carene is important. At present, the low activity of 3-carene synthase (CarTS) and the insufficient supply of precursors in Saccharomyces cerevisiae significantly constrain the synthesis of 3-carene. In this study, the NPP orthogonal pathway, source screening, CarTS modification, enzymatic subcellular localization, and molecular chaperone screening were used to promote 3-carene synthesis. By introducing the NPP orthogonal pathway and truncated CarTS from Salvia stenophylla (SsCarTS), the yield of 3-carene increased by 32.1-fold. Subsequently, through enzymatic subcellular localization and molecular chaperone screening, the yield of 3-carene was increased by 39.0%, reaching 50.6 mg/L in shake flask cultures. Eventually, the final titer of 3-carene reached 506.7 mg/L in a 5-L bioreactor by optimizing the culture medium containing metal ions and vitamins, which is the highest reported titer of 3-carene in microbial cell factories. In this study, we provide a cell factory for the synthesis of 3-carene as well as reference data for the biosynthesis of other bicyclic monoterpenes.

The ethylene-forming enzyme (EFE) is a member of the mononuclear non-heme Fe(II)- and 2-oxoglutarate-dependent oxygenase superfamily, which can oxidize 2-oxoglutarate to form ethylene in an arginine-dependent reaction. While significant enzyme engineering efforts have targeted the active site and surface of EFE, to date, no variant with substantially improved activity has been reported. To enhance catalytic activity and broaden the application potential of EFE, this study developed a surface engineering strategy based on structural analysis and potentially new l-Arg binding information. The resulting variant, E213T, exhibited a 1.5-fold increase in catalytic activity and a 2.4-fold elevation in k_cat, l-Arg. Molecular dynamics simulations further revealed that this amino acid substitution reduced the affinity of the surface l-Arg binding site and altered the accessibility of ligands to the catalytic center. Our study provides a new perspective on the distal sites and functional relationships in protein engineering of EFE.

Microsporidia are obligate intracellular fungal parasites that are widely distributed in natural and agricultural environments and represent an important biological hazard to economically important insects. Nosema bombycis (N. bombycis), the causative agent of pébrine disease, poses a serious threat to silkworm breeding and sericultural sustainability. However, the molecular mechanisms underlying microsporidian proliferation and host-pathogen interactions remain poorly understood. In this study, we systematically characterized the long noncoding RNAs (lncRNAs) and microRNA-like RNAs (milRNAs) of N. bombycis, an important pathogen of Bombyx mori (B. mori). Using the full-length transcriptome and small RNA sequencing data from B. mori midgut tissues infected with N. bombycis at key developmental stages, we identified 30 novel lncRNAs and 12 previously unreported milRNAs in N. bombycis. We further established a competing endogenous RNA regulatory network involving N. bombycis-derived lncRNAs, parasite-specific milRNAs, and host-derived microRNAs (miRNAs), revealing intricate molecular interactions that may be implicated in pathogenicity. Notably, NbLNC2914, a highly expressed lncRNA, was shown to function as a molecular sponge for the host miRNA bmo-miR-2808a-3p, modulating its expression and consequently regulating the downstream N. bombycis gene, NBO_58g0005. Functional validation assays demonstrated that this regulatory axis had an important impact on N. bombycis proliferation. Our findings reveal a previously unrecognized noncoding RNA (ncRNA)-mediated regulatory axis that facilitates N. bombycis proliferation within host cells. This study provides mechanistic insights into the molecular basis of microsporidia pathogenicity and highlights ncRNAs as potential molecular targets for mitigating microsporidia-associated biological hazards in agricultural ecosystems.

Modification of the gut microbiota by beneficial microbes can enhance an organism’s lifespan, giving rise to the concept of probiotics. Probiotics are live microorganisms that provide health benefits when taken in sufficient amounts. Owing to their outstanding health benefits, probiotics have experienced rapid expansion and gained interest for the development of new applications. The exploration of microbial applications via genetic modification is currently of great interest to researchers. Genetic engineering using the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas system has received considerable attention and has established applications. Owing to these enhanced properties, the CRISPR-Cas system is currently used in medicine, agriculture, food, and biotechnology. Considering the adaptive immune system in bacteria, this genetic tool is used to alter the microbial genome. Lactic acid bacteria (LAB) are widely recognized for their probiotic potential, and over 40% of LAB species contain the CRISPR-Cas system. The rising demand for probiotics and their expanding applications necessitate the enhancement of their existing characteristics. The CRISPR-Cas system, recognized for its precision, accuracy, and speed, has enabled researchers to modify the genomes of probiotics, thereby enhancing their beneficial attributes. This system can enhance probiotic properties through additive, subtractive, or modulatory mechanisms. Various approaches have been developed to improve probiotic functionalities using the CRISPR-Cas system, such as substituting slow promoters with efficient alternatives, eliminating undesirable components, boosting metabolism, and increasing tolerance levels. Furthermore, CRISPR-engineered probiotics have emerged as next-generation probiotics with enhanced properties and advanced applications across diverse fields, including the food, medicine, agriculture, and pharmaceutical sectors.

Hydrogenotrophic methanogens are promising biocatalysts for biomethanation and carbon dioxide utilization, yet their robustness under bioprocess perturbations remains insufficiently defined. We quantified robustness in a Methanothermobacter archaeal strain with proven potential for industrial application across temperature shifts, oxidative exposure, nitrogen depletion, and hydrogen starvation, measured five key cellular functions in batch culture, and derived a Fano factor based robustness metric that links data dispersion to functional stability. Thermal and oxidative stress were the primary constraints on robustness, most notably for methane productivity and lag phase duration, whereas hydrogen starvation increased productivity in some cases without large losses in robustness, and nitrogen depletion had limited effects. Global proteomics revealed coordinated changes consistent with these patterns, including increased ribosomal proteins, trehalose synthesis, chaperones, and redox regulators under thermal stress, and enrichment of PAS and histidine kinase domain proteins under oxidative stress. Structure-guided predictions using Alpha Fold 3 supported the hypothesis that stress responsive proteins may associate with canonical methanogenesis core subunits, as FmdE was predicted to associate with FmdE-like paralogs under thermal stress, while several Mtr subunits decreased in abundance. The combined results identify actionable targets for engineering robustness in methanogenic archaea, including stabilizing multi subunit methanogenesis complexes such as Mtr, tuning PAS and HK sensors to improve redox response, and modulating chaperone and osmoprotection capacity to regulate metabolic functions during temperature fluctuations. As derived future work, mapping protein interactions with abundance profiling may help move proteomics from description to prediction, providing network-informed design rules that complement conventional genome-centered proteomics and guide strain optimization of robust archaeal biocatalysts for biomethanation.

Dynamic regulatory systems are promising tools for the biosynthesis of high-value chemicals. However, the construction of pathway-independent multimodule self-induced regulatory systems capable of modulating complex metabolic pathways remains challenging. In this study, we developed a multimodule self-induced regulatory system based on two orthogonal quorum sensing (QS) and a stationary-phase sensing system. We optimized the Las and Tra^∗ QS response times by tuning acyl-homoserine lactone (AHL) synthase expression, identified a stationary-phase sensor with a higher output, and integrated these into a pathway-independent multimodule self-induced regulatory system. This system successfully enabled the sequential expression of multiple genes at specified time intervals under autoinduction conditions. Ultimately, this system, combined with a small RNA inhibition module, was employed to produce 3-hydroxypropionic acid (3-HP), which sequentially regulated the three modules of the tricarboxylic acid cycle, fatty acid metabolism, and 3-HP synthesis. Application of this system resulted in a 3-HP titer of 7.0 g/L in shake flask fermentation, which was 27% higher than that of the base strain using a static regulatory strategy, while also improving bacterial growth. This application validates the potential of the developed multimodule self-induced regulatory system to regulate multigene expression in complex microbial metabolic networks.

Crude oil production has been enhanced using microbial-enhanced oil recovery (MEOR) in many oilfields. After over six years of MEOR test and field application in high-salt environments and unconventional oil reservoirs of plateau oilfields, MEOR mechanisms of emulsification, surface tension reduction, petroleum degradation, and reservoir microbial community structure adjustment were developed. Based on the core MEOR mechanisms of microbes, we carried out paraffin wax removal, single-well huff-puff, and microbial flooding in the Qinghai oilfield, thereby increasing oil production and sustaining economic benefits. These results demonstrate that microorganisms can be enhanced and applied in oilfields with large temperature differences, high salinity, and unconventional reservoirs.

Current research on microbial salinity adaptation faces substantial challenges, including the limited predictive accuracy of traditional single-gene models and difficulty in dissecting systemic biological responses to salinity stress in complex natural habitats. To overcome these bottlenecks, the multi-model ensemble learning tool SuSha, which leverages genome-wide amino acid composition features, was developed. By extracting features from the whole-genome data of 123 bacterial and archaeal species with well-defined salinity adaptations, a 24-dimensional feature vector was constructed, comprising the frequencies of 20 standard amino acids and four aggregated functional categories. Based on this, an ensemble model was developed by integrating algorithms such as random forest, bagging, and extra trees. Five-fold cross-validation demonstrated that this 24-dimensional feature-based ensemble model achieved a global accuracy of 0.765 and an area under the curve of 0.941, significantly outperforming individual baseline models. Furthermore, the model was externally validated using 2678 metagenomic samples from six global regions, encompassing freshwater, marine, and hypersaline habitats. SuSha exhibited high robustness, ecological consistency across diverse salinity gradients, and a classification accuracy of over 90% for extreme halophiles, particularly within the extreme halophilic range. By enabling high-precision genotype-to-phenotype predictions using a habitat-adaptive algorithm-switching strategy, SuSha provides a robust computational framework for inferring the physiological potential of uncultivated microorganisms and mining microbial resources in extreme environments.

Conventional activated sludge processes are primarily designed for nitrogen and phosphorus removal, with carbon transformation regarded as a concomitant process that supports downstream denitrification rather than a proactively regulated process. Inspired by the metabolic division of labor in gut ecosystems, we proposed and validated a metabolism-guided strategy for an experimental membrane bioreactor (MBR-E) with a prefermentation unit that couples upstream fermentation with downstream denitrification to restructure carbon flux toward more bioavailable electron donors for nitrogen removal. Long-term operation showed that MBR-E achieved significantly lower effluent total nitrogen (7.9 ± 2.4 mg/L) compared with the control MBR system (MBR-C, 12.3 ± 3.5 mg/L), which was attributed to its elevated specific denitrification rate. Influent organics were efficiently converted into volatile fatty acids (VFAs) via fermentation and shortening hydraulic retention time from 0.67 h to 0.5 h shifted VFA composition from propionate/butyrate dominance to acetate enrichment. Further, 16S rRNA gene sequencing demonstrated that functional denitrifiers and nitrifiers were selectively enriched in MBR-E. Co-occurrence network analysis revealed strengthened cooperative interactions and tighter functional coupling between carbon degradation and nitrogen removal in MBR-E. Overall, this study demonstrates that fermentation-driven carbon reprogramming can effectively regulate downstream respiratory pathways and reshape the microbial community structure, providing a novel approach for efficient nitrogen removal from low-carbon/nitrogen wastewater.