Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a class of secondary metabolites synthesized by the ribosomes of microorganisms and formed through a series of post-translational modifications. They have diverse structures, high stability, and antimicrobial, antiviral, anti-inflammatory or anti-tumor activities. Moreover, they are not prone to generating drug resistance, thus showcasing great potential to be applied in the fields of medicine, food, and agriculture. The genomes of microorganisms harbor a large number of biosynthetic gene clusters for RiPPs, while many of them require the producing strains to be cultivated in specific conditions or to interact with other environmental microorganisms, being the “dark matter” in the genome. Heterologous biosynthesis is an effective means to obtain novel RiPPs and make use of them. This article reviews the recent research progress in the diversity, bioactivity, genomic mining, and heterologous biosynthesis of RiPPs from microorganisms, with the expectation of providing a theoretical basis for a deeper understanding of the molecular structures and functions of RiPPs, as well as for the development and application of novel microbial active metabolites and their producers.

Microorganisms represent the largest untapped resource reservoir on the Earth, and breakthroughs in their isolation and cultivation are prerequisites for fundamental advances in the life sciences. This review focuses on recent progress in the isolation and screening technologies for bacteria, fungi, and archaea. It systematically elucidates how the development and application of cutting-edge isolation and screening technologies have enhanced the efficiency of isolating previously uncultivable and rare microbial taxa. By summarizing lineage-specific strategies—such as multi-omics targeting and single-cell precision localization for bacteria, metabolomics-guided screening and microfluidic technology for fungi, and co-culture systems coupled with extreme-condition cultivation for archaea—this review highlights the core value of interdisciplinary technology integration in bridging genomic data with in situ functional validation. Finally, the article prospectively addresses challenges in data integration and the construction of automated workflows, thereby outlining a strategic pathway for the systematic exploration of microbial resources.

Objective The engineering of the reductive glycine pathway (rGlyP) in Komagataella phaffii (syn. Pichia pastoris) represents a promising strategy for the co-utilization of methanol and CO₂. However, the efficiency of this pathway is constrained by the insufficient supply of intracellular reduced nicotinamide adenine dinucleotide (NADH), as the native alcohol oxidase (AOX) pathway generates hydrogen peroxide rather than NADH, leading to energy loss and oxidative stress. To overcome this bottleneck, this study reconstructed the methanol oxidation pathway and employed a subcellular compartmentalization strategy to optimize the carbon flux and energy metabolism. Methods Five different sources of NAD⁺-dependent methanol dehydrogenase (MDH) were screened in an aox1/aox2-deficient strain by using the growth curve and methanol utilization rate as indicators to determine the optimal MDH, and the methanol induction concentration was optimized. Subsequently, a compartmentalization strategy was employed by fusing the peroxisomal targeting signal 1 (PTS1) to MDHN1T, which targeted the enzyme to the peroxisome to spatially couple methanol oxidation with formaldehyde detoxification. Results The MDHN1T derived from Cupriavidus necator had the best catalytic performance, and the optimum methanol induction concentration was optimized to be 0.6%. Under co-utilization conditions, the engineered strain achieved a methanol consumption rate of 28.98 mg/d, with the total intracellular NAD_total pool, NADH/NAD⁺ ratio, and biomass being 1.3, 1.2, and 2.2 folds, respectively, of those in the parental strain. Conclusion This study successfully alleviates the redox cofactor imbalance in the rGlyP and enhances co-utilization of methanol and CO₂ in K. phaffii, providing a robust chassis and a theoretical basis for the development of microbial cell factories utilizing one-carbon resources.

Objective Four-carbon dicarboxylic acids are a class of important platform chemicals widely used in the food, pharmaceutical, and chemical industries. However, the efficiency of microbial fermentation for producing four-carbon dicarboxylic acids still faces challenges, mainly limited by insufficient central carbon metabolic flux and byproduct accumulation. Methods This study used Escherichia coli as the chassis strain and adopted a strategy combining rational metabolic engineering and non-rational modification to systematically optimize the four-carbon dicarboxylic acid synthesis capacity of E. coli. Results The non-cyclic glyoxylate shunt was reconstructed and the expression of key pathway enzymes was optimized to enhance the metabolic flux toward four-carbon dicarboxylic acids. The synthesis capacity of four-carbon dicarboxylic acids was enhanced by employing atmospheric and room-temperature plasma (ARTP) mutagenesis. The knockout of key genes in the acetate, formate, and lactate synthesis pathways effectively minimized carbon flux diversion, thereby enhancing the availability of oxaloacetate, the central precursor to four-carbon dicarboxylic acids. On this basis, through specific modification of terminal metabolic pathways, the engineering strain E. coli Fum02 for fumaric acid production were constructed. Finally, in a 5 L fermenter, the fumaric acid titer, yield, and productivity of the engineering strain E. coli Fum02 reached 45.2 g/L, 0.45 g/g, and 0.23 g/(L·h), respectively. Furthermore, by blocking the succinate dehydrogenase gene (sdhAB) and implementing fermentation optimization strategies, this platform strain could also be redirected toward efficient succinate production. Conclusion This study provides a reference for the metabolic engineering modification of bacteria to produce organic acids and also lays a foundation for the industrial biomanufacturing of four-carbon dicarboxylic acids.

Objective The rapid increase in wastewater discharge from animal husbandry has caused severe environmental pollution. Identifying efficient heterotrophic nitrifying-aerobic denitrifying bacteria and investigating their denitrification mechanisms are of great theoretical and practical importance for mitigating nitrogen pollution in the wastewater. Methods A strain exceling in heterotrophic nitrification-aerobic denitrification (HN-AD) was isolated and from activated sludge in pig farms. Culture conditions were optimized by response surface methodology. We evaluated the inorganic nitrogen-transforming capacity of the strain by assessing its utilization efficiency of single and mixed nitrogen sources and through nitrogen balance analysis. The completeness of the denitrification process was confirmed via gas chromatographic measurements of N₂ and N₂O. Finally, the nitrogen removal pathways and underlying mechanisms were elucidated through whole-genome analysis. Results The successfully isolated strain Klebsiella sp. WH-E exhibited excellent HN-AD capabilities. The growth conditions of the strain were optimized as follows: sodium citrate as the carbon source, 34.18 °C, initial pH 7.1, a C/N ratio of 14.53, and a shaking speed of 159.59 r/min. When the strain was cultured with ammonium, nitrate, or nitrite as the sole nitrogen source, the nitrogen removal rates were 99.80%, 81.54%, and 80.00%, respectively. Furthermore, when ammonium was the sole nitrogen source, 35.84% and 35.91% of nitrogen were converted into cellular nitrogen and gaseous nitrogen, respectively. When ammonia nitrogen was combined with nitrate nitrogen as mixed nitrogen sources, the nitrogen removal rate was 100.00%; When ammonia nitrogen was combined with nitrite nitrogen as mixed nitrogen sources, the ammonia nitrogen removal rate was 100.00%, and the nitrite nitrogen removal rate was 91.97%, respectively. Whole-genome sequencing identified several nitrogen metabolism-related functional genes, including glnB, norVWR, narGHI, nasBC, and nirBD. Conclusion Klebsiella sp. WH-E possesses three nitrogen metabolism pathways: ammonium assimilation, nitrification-denitrification, and nitrate assimilation and dissimilation. This study confirms the applicability of Klebsiella sp. WH-E for nitrogen removal from full-scale piggery wastewater and establishes a solid theoretical foundation for its engineering applications.

The gut microbiota plays a crucial role in promoting food digestion in animals. However, the impact of cross-species microbiota transplantation from donors with different dietary habits on the host food digestion capacity remains unclear. Objective To investigate the role of cross-species microbiota transplantation in regulating the digestive system adaptability, metabolic functions, reproduction, stress responses, and gut microbiota structure of the host. Methods We utilized New Zealand white rabbits (Oryctolagus cuniculus), a herbivorous species, and C57BL/6J mice, an omnivorous species, as donors and recipients of gut microbiota, respectively. The mice were allocated into three groups: a control group on a normal diet (Con), a group on a high-fiber diet (TS), and a group on a high-fiber diet supplemented with rabbit fecal microbiota transplantation (OC). This study was designed to evaluate various physiological and biochemical parameters, including body weight, food intake, absolute and relative organ weights (both wet weight and organ-to-body weight ratio), morphometric indices (length and diameter) of the small intestine, sperm concentration, and serum corticosterone level, in mice. Additionally, we performed 16S rRNA gene sequencing targeting the V3-V4 hypervariable region to characterize the composition of fecal microbiota. Results A high-fiber diet significantly increased the food intake, small intestine length, and serum corticosterone level, while significantly reducing the body weight, liver and spleen wet weights, liver/body weight ratio, spleen/body weight ratio, and sperm concentration in mice. Moreover, it increased the alpha diversity of the gut microbiota, decreased the Bacillota-to-Bacteroidota ratio, and reduced the relative abundance of probiotics (such as Ligilactobacillus). Transplantation of the gut microbiota from rabbits increased the wet weight of the epididymis and the epididymis/body weight ratio, while significantly reducing the liver/body weight ratio and the serum corticosterone level in recipient mice. Furthermore, a high-fiber diet significantly increased the relative abundance of the fiber-degrading bacterial family (Oscillospiraceae) and the gut health-associated bacterial genus (Colidextribacter). After the transplantation of rabbit gut microbiota into mice, the relative abundance of Oscillospiraceae and Colidextribacter in mice increased significantly. Conclusion The high-fiber diet has adverse effects on omnivores. Although the microbiota transplantation from herbivores does not significantly improve the host ability to digest fiber, it changes the gut microbiota structure of omnivores, playing a positive role in improving their digestion, reproduction, metabolism, and stress responses. Future research needs to further determine the optimal levels of dietary fiber for omnivores and the dosage of microbiota transplantation from herbivores, as well as their synergistic effects and underlying mechanisms in improving animal health. This study provides a reference for exploring the role of gut microbiota in animal adaptation to dietary changes in natural environments and lays a foundation for future research on improving the utilization of high-fiber foods by omnivorous domestic animals.

Objective To explore plant growth promoting rhizobacteria (PGPR) resources from the rhizosphere soil of maize in a maize-soybean rotation system and elucidate their roles in promoting the growth of maize and soybean, thus providing a theoretical basis and practical support at the microbial level for the sustainable development of agriculture. Methods Actinomycetes strains were isolated from the rhizosphere soil of maize via the dilution plating method with Gauze’s Synthetic Medium No. 1. The strains capable of secreting protease, producing siderophores, and fixing nitrogen were selected out. The isolated strains were identified by means of morphological observation and 16S rRNA gene sequence analysis. After optimization of the fermentation conditions and tests of stress tolerance, a synthetic microbial consortium (SMC) was prepared. Its growth-promoting effects on maize and soybean were evaluated through seed germination tests and pot experiments. Results A total of 105 Actinomycetes strains were isolated, five of which simultaneously exhibited the abilities of siderophore production, protease secretion, and nitrogen fixation. These strains were identified as Arthrobacter pokkalii (JM-18, JM-21), A. oryzae (JM-24), A. ginsengisoli (JM-47), and A. pascens (JM-48). They were mixed in equal proportions to form a SMC. Growth promotion assays showed that the SMC significantly improved maize seed germination and maize plant growth in pots. Specifically, the SMC increased the root length and shoot length in the seed germination assay by 120.22% and 20.94%, respectively, and it also significantly increased the plant height, root length, fresh weight, and dry weight of maize plants in pots. Moreover, the SMC markedly promoted soybean development, increasing soybean shoot length by 42.08% during seed germination. For potted soybean plants, the SMC increased the plant height, root length, fresh weight, and dry weight by 39.40%, 93.31%, 161.14%, and 163.57%, respectively. Conclusion We successfully identified five Actinomycetes strains capable of secreting protease, producing siderophores, and fixing nitrogen. The SMC constructed from these strains significantly enhances the growth of both maize and soybean. This study offers promising microbial resources for the development of efficient and environmentally friendly biofertilizers.

Objective With the widespread use and promotion of plastic film mulching on the Qinghai-Xizang Plateau, a series of issues caused by its application have also emerged. Given the fragile eco-environment of the plateau, it is necessary to investigate the effects of different types of plastic film mulching on the soil microbial community structures in farmland ecosystems. Methods Three treatments—pre-planting soil (ZQ), soil covered with conventional polyethylene mulch (CMPs), and soil covered with biodegradable mulch (BMPs)—were established. Soil physicochemical properties were measured, and high-throughput sequencing of the 16S rRNA gene and ITS region was employed to analyze microbial diversity, community structure, and their associations with environmental factors, on the basis of which the impacts of mulch types on soil microorganisms were evaluated. Results Differences in soil physicochemical factors were observed among different treatments (P<0.05). There were no significant differences in alpha diversity indices for both bacteria and fungi among the treatments, indicating that short-term plastic film mulching did not significantly alter the richness and diversity of microbial communities. The dominant bacterial phyla were Pseudomonadota, Actinomycetota, Acidobacteriota, and Chloroflexota, with most dominant genera being unclassified. The dominant fungal phyla were Ascomycota, Basidiomycota, Mortierellomycota, with dominant genera including Mortierella and Solicoccozyma. Network analysis revealed that the main drivers of bacterial and fungal community structures were pH and microplastic (MP) content, respectively. This result reflected functional differences of fungi and bacteria. Fungi, as primary decomposers, were more sensitive to MP pollution, whereas bacterial community structure was more closely related to soil pH. Functional prediction showed that, in bacteria, only the metabolism pathway within the KEGG level 1 showed a positive correlation with the mulching treatment, and no significant differences in COG functions were observed between treatments. In fungi, saprotrophic functions predominated, and their relative abundance changed significantly among treatments. Conclusion Short-term plastic mulching does not significantly affect microbial alpha diversity, but alters the community structure. Compared with conventional PE mulch, biodegradable mulch shows greater potential in enhancing soil nitrogen and organic carbon pools. However, it leads to more severe short-term MP accumulation, accompanied by the risk of pathogenic fungal enrichment. Therefore, its long-term ecological effects require further assessment.

Trees can form mutualistic symbionts with mycorrhizal fungi. Different mycorrhizal types affect the community structure of endophytic fungi by regulating tree physiology and root microenvironment, thus becoming a key link driving the interaction network between soil and microorganisms in tropical forests. However, the mechanisms by which different mycorrhizal types regulate the diversity and community composition of endophytic fungi in tropical tree roots are still not fully understood. Objective To explore the effects of different mycorrhizal types on the diversity and community structure of root endophytic fungi in tropical trees, as well as their key driving factors, systematically clarifying how mycorrhizal types affect the composition and diversity of endophytic fungal communities by regulating root traits and rhizosphere environment, and identifying the key driving factors. Methods On the basis of 3 773 sets of soil and root data collected from three research sites of Chinese Ecosystem Research Network (CERN) in Xishuangbanna tropical forest, China, we integrated and constructed a dataset at the tree species level. This dataset encompassed data of the root traits, soil physical and chemical properties, and the operational taxonomic unit (OTU) abundance of endophytic fungi in the roots of 119 trees (54 species) with arbuscular mycorrhizas (AM) and 31 trees (12 species) with ectomycorrhizas (ECM), and it was then used for the research. Results The alpha diversity of endophytic fungi in the roots of AM trees was higher than that of ECM trees (P<0.05). Mycorrhizal types affected the dominant groups of root endophytic fungi. Ascomycota had the highest relative abundance (43.17%) in the roots of AM trees, and Basidiomycota had the highest relative abundance (65.17%) in the roots of ECM trees. The co-occurrence network analysis showed that the endophytic fungal network was denser in the roots of AM trees and more modular in roots of ECM trees. Soil properties were the dominant driving factors for the endophytic fungal communities in the roots of AM trees, while the endophytic fungal communities in the roots of ECM trees were regulated jointly by root traits and soil properties. Soil phosphorus was a key factor affecting the endophytic fungal communities in the roots of AM and ECM trees. Conclusion In tropical forest ecosystems, AM drives trees to form species-rich and closely interacting endophytic fungal communities in the roots, and the assembly process is mainly regulated by soil factors. ECM trees form a specialized symbiotic fungal system, whose construction is regulated by both root traits and soil factors. In addition, soil phosphorus is the core factor driving the formation of endophytic fungal communities in the roots of the two types of trees.

Objective Fusarium wilt caused by Fusariumoxysporum f. sp. nivum is a typical soil-borne disease in watermelon production, posing significant threats. This study investigates the microbial community structures in the rhizosphere soil of healthy and Fusarium wilt-affected watermelon plants to clarify the regulatory effects of this disease on the physicochemical properties and microbial communities of rhizosphere soil. It aims to reveal the interactions between pathogen enrichment, beneficial microbial decline, and soil environmental factors, providing theoretical support for the green control of Fusarium wilt in watermelon plants by rhizosphere microbiome regulation. Methods Rhizosphere soil samples were collected from healthy plants (HT group) and Fusarium wilt-infected plants (FT group) of the watermelon variety ‘Xiaoyu No. 5’ in Shaoyang, Hunan. Physicochemical indicators including total nitrogen (TN), total phosphorus (TP), available phosphorus (AP), and available potassium (AK) were measured. Illumina high-throughput sequencing was employed to analyze the structures and diversity of microbial communities in the rhizosphere soil of healthy and disease-infected plants. Results The FT group had lower content of TP, AP, and AK in the rhizosphere soil than the HT group (P<0.05). The TN, organic matter (OM), and pH in the FT group were lower without significant differences than the HT group. The FT group had higher fungal ACE and Chao1 indices (P<0.05), higher bacterial ACE and Chao1 indices (P>0.05), and higher fungal and bacterial Simpson indices (evenness) (P<0.05) than the HT group. The abundance of Bacillota was significantly higher in the HT group than in the FT group, whereas that of Ascomycota was significantly higher in the FT group. At the genus level, the abundance of beneficial bacteria such as Neobacillus and Bacillus decreased in the FT group, while that of the pathogenic genus Fusarium increased sharply from 0.06% to 2.40%. The redundancy analysis (RDA) indicated that TN, TP, and OM were key drivers of bacterial community changes, whereas TN, OM, and AK were core regulators of fungal communities. Functional prediction suggested enhanced functions such as stress responses and energy metabolism of bacteria, alongside increased potential for functions such as plant cell wall degradation of fungi, in the diseased rhizosphere. Conclusion The occurrence of Fusarium wilt in watermelon plants leads to depletion of phosphorus and potassium in the rhizosphere soil and disrupts microbiome balance. This is manifested by the enrichment of Fusarium and the decline of beneficial bacteria (e.g., Neobacillus and Bacillus). Soil TN, OM, and AK are key environmental factors regulating this imbalance, with AK deficiency potentially serving as a pivotal link between soil environmental degradation and disease intensification. These findings provide crucial theoretical support for developing eco-friendly control strategies-potassium supplementation and stabilization alongside the targeted cultivation of beneficial microbial communities-targeting Fusarium wilt in watermelon plants.