The rare biosphere harbors immense microbial diversity, yet most low-abundance taxa remain uncultured and functionally enigmatic. Here, we isolated strain D14T from deep-sea water, and propose to classify it as a novel species, Metabolovarius oceani sp. nov., within the novel family Metabolovariaceae fam. nov. M. oceani represents the first cultivated member of the candidate family NORP267, a globally distributed but elusive alphaproteobacterial lineage known only from metagenome-assembled genomes. It possesses broad metabolic capabilities, including CO2 fixation, polyhydroxyalkanoate biosynthesis, complete denitrification and thiosulfate oxidation, and is capable of aerobic growth under both heterotrophic and autotrophic conditions and of anaerobic autotrophic denitrification via thiosulfate oxidation. Despite its versatile metabolic repertoire and global distribution, Metabolovariaceae remains consistently low in abundance across diverse habitats. The isolation of M. oceani permits direct experimental insights into the evolutionary adaptations, physiological resilience, and potential ecosystem roles of rare but metabolically versatile microorganisms within the microbial dark matter.

Understanding microbial adaptation to extreme environments remains a key challenge in microbial ecology. Geothermal hot springs, characterized by temperature gradients and varying geochemical conditions, represent valuable natural laboratories for studying microbial diversity, adaptive strategies, and evolutionary mechanisms. However, despite many studies of hot spring communities, how temperature gradients shape key microbial adaptation strategies remains insufficiently understood, limiting our ability to explain survival and function in extreme environments. Our study investigated microbial community composition and functional profiles across a natural thermal gradient (50–93 °C) in six hot springs on the Western Sichuan Plateau using optimized contig- and MAG-based metagenomic strategies. Enhanced annotation approaches significantly improved taxonomic resolution in these extreme environments. Metagenomic analyses revealed distinct shifts in microbial communities along the thermal gradient: moderate-temperature springs (50–70 °C) were dominated by Pseudomonadota and Bacteroidota, exhibiting heterotrophic flexibility and utilizing the Calvin–Benson–Bassham cycle and diverse nitrogen reduction pathways; high-temperature springs (70–90 °C) were enriched in Chloroflexota, which primarily employed the Wood–Ljungdahl pathway coupled with enhanced sulfur metabolism; and extreme-temperature springs (≥ 90 °C) were characterized by Aquificota and Thermoproteota, relying on specialized autotrophic pathways (rTCA, DH/HH cycles), streamlined nitrogen assimilation, and sulfur oxidation pathways. These thermophilic lineages showed genome streamlining, reduced regulatory complexity, and specialized metabolic strategies, reflecting narrower ecological niches and deeper phylogenetic branches. This metagenomic investigation across a temperature gradient in western Sichuan hot springs highlights temperature as an essential driver of microbial community structure, genome evolution, and adaptive specialization. Thermophilic lineages in extreme-temperature environments exhibited streamlined genomes, specialized metabolic functions, and narrower ecological niches, consistent with adaptation to persistent thermal stress. The findings enhance understanding of microbial evolutionary strategies and underscore the ecological significance of temperature-driven adaptation in extreme environments. Temperature-driven microbial community structure shifts across thermal gradients. Thermophiles in hotter springs show deeper phylogeny and narrower niche breadth. Carbon fixation transition from rTCA, DH, HH to 3H, CBB along thermal gradients. Genome Streamlining and GC/amino acid shifts enhance thermal structural stability. Composite mapping strategy improves metagenome annotation in extreme environments.

Nicotinamide mononucleotide (NMN), a direct precursor of the essential coenzyme nicotinamide adenine dinucleotide (NAD+), confers anti-aging effects and multiple health benefits. Engineered microorganisms represent a promising platform for sustainable industrial production of NMN. Here, the previously reported NMN-producing strain NMN008 was engineered to co-utilize glucose and glycerol for the biosynthesis of NMN from nicotinamide (NAM). First, the glycolytic genes pgi and pykA/pykF were sequentially deleted to disrupt glucose catabolism through the glycolytic pathway, thereby potentially improving precursor availability for NMN biosynthesis. Second, a feedback-resistant glycerol kinase mutant (glpK*) was introduced to enhance glycerol utilization, aiming to compensate for the growth defects associated with impaired glycolysis. These modifications enabled glycerol to primarily support cell growth and energy metabolism, while improving glucose allocation toward NMN biosynthesis by reducing its competitive consumption through glycolysis. As a result, the final strain achieved an NMN titer of 32.92 g/L in a 2 L bioreactor, representing a 26.28% increase in NMN production and a substantial 34.48% improvement in carbon conversion efficiency. Our research provides an effective strategy to achieve industrial-scale production of NMN, laying a foundation for the widespread application of NMN.

Fast-growing Vibrio natriegens is now recognized as a next-generation chassis for synthetic biology and biotechnology; however, its low transformation efficiency, limited gene editing methods and high fermentation cost are still the main challenges hampering its industrial application. In this study, we established an efficient electroporation transformation and dual-plasmid CRISPR-Cas9 editing system in V. natriegens. Subsequently, the heterologous ergothioneine biosynthetic pathway involving the combination of the superstrong PLlacO1 promoter and weak RBS7 was constructed in V. natriegens. Multiple genes encoding genes involved in byproduct formation and adenosine triphosphate (ATP) degradation were consecutively deleted, while several key genes involved in the S-adenosylmethionine (SAM) cycle and the ATP synthesis pathway were overexpressed to increase ergothioneine production. Finally, fed-batch fermentation was performed using low-cost sucrose as the sole carbon source under high-salinity, non-sterile conditions, resulting in an ergothioneine titer of 1.2 g/L in a 2-L bioreactor. This study not only provides the first successful example of the ergothioneine biosynthesis with engineered V. natriegens strains but also establishes an efficient and economic platform in which V. natriegens is used to produce other high-value compounds.

Nicotinamide mononucleotide (NMN), a key precursor of NAD+, is a promising nutraceutical due to its excellent efficacy in alleviating aging and disease. The bioproduction of NMN faces challenges related to incomplete metabolic engineering and insufficient metabolic flux. Here, we constructed an NMN synthesis pathway in Escherichia coli BW25113 by deleting the competitive pathway genes and introducing three heterologous genes encoding the key enzymes nicotinamide phosphoribosyltransferase (NAMPT), phosphoribosyl pyrophosphate synthetase and an NMN transporter. Next, the identification of a highly active NAMPT and optimization of gene expression markedly increased the conversion of NAM to NMN, with a titer of 3503.85 mg/L in shake flasks. Furthermore, by facilitating the coutilization of glucose and xylose, more metabolic flux was diverted toward PRPP biosynthesis, resulting in an NMN titer of 15.66 g/L through whole-cell catalysis and 46.66 g/L in a 2-L bioreactor. This represents the highest NMN yield reported to date, exhibiting great potential for initiating sustainable industrial production of NMN.

Bacterial persisters, a subpopulation of genetically susceptible cells that are normally dormant and tolerant to bactericides, have been studied extensively because of their clinical importance. In comparison, much less is known about the determinants underlying fungicide-tolerant fungal persister formation in vivo. Here, we report that during mouse lung infection, Cryptococcus neoformans forms persisters that are highly tolerant to amphotericin B (AmB), the standard of care for treating cryptococcosis. By exploring stationary-phase indicator molecules and developing single-cell tracking strategies, we show that in the lung, AmB persisters are enriched in cryptococcal cells that abundantly produce stationary-phase molecules. The antioxidant ergothioneine plays a specific and key role in AmB persistence, which is conserved in phylogenetically distant fungi. Furthermore, the antidepressant sertraline (SRT) shows potent activity specifically against cryptococcal AmB persisters. Our results provide evidence for and the determinant of AmB-tolerant persister formation in pulmonary cryptococcosis, which has potential clinical significance.

Background Prolyl endopeptidase from Aspergillus niger (AN-PEP) is a prominent serine proteinase with various potential applications in the food and pharmaceutical industries. However, the availability of efficient and low-cost AN-PEP remains a challenge owing to its low yield and high fermentation cost. Results Here, AN-PEP was recombinantly expressed in Trichoderma reesei (rAN-PEP) under the control of the cbh1 promoter and its secretion signal. After 4 days of shaking flask cultivation with the model cellulose Avicel PH101 as the sole carbon source, the extracellular prolyl endopeptidase activity reached up to 16.148 U/mL, which is the highest titer reported to date and the secretion of the enzyme is faster in T. reesei than in other eukaryotic expression systems including A. niger and Komagataella phaffii . Most importantly, when cultivated on the low-cost agricultural residue corn cob, the recombinant strain was found to secret a remarkable amount of rAN-PEP (37.125 U/mL) that is twice the activity under the pure cellulose condition. Furthermore, treatment with rAN-PEP during beer brewing lowered the content of gluten below the ELISA kit detection limit (< 10 mg/kg) and thereby, reduced turbidity, which would be beneficial for improving the non-biological stability of beer. Conclusion Our research provides a promising approach for industrial production of AN-PEP and other enzymes (proteins) from renewable lignocellulosic biomass, which provides a new idea with relevant researchers for the utilization of agricultural residues.

Cold stress influences the growth and geographical distribution of plants. Tobacco (Nicotiana tabacum L.), a model in plant biology studies and a world-widely cultivated economic plant from tropical America, exhibits extreme sensitivity to low temperatures. The conserved B-BOX (BBX) transcription factors play vital roles in plant photomorphogenesis and flowering regulation, and their functions in abiotic stresses, including cold stress, have been uncovered recently. However, few studies on tobacco BBX genes have been conducted to date. In this study, 49 non-redundant BBX genes have been identified in the reference tobacco genome. Gene structure, phylogenetic relationship, prediction of cis-regulatory elements in promoters, tissue-specific expression patterns, and response to cold stress were conducted on these BBX gene members. The expression of NtBBX9 and NtBBX11, two close members in the amplified group II, was up-regulated under cold treatments. Overexpression of NtBBX11 embedded the cold tolerance of transgenic plants, while the NtBBX9 overexpressors were susceptible to cold. The NtBBX9 functions as a negative regulator of tobacco response to cold stress, which was confirmed by the phenotype of NtBBX9 RNA-silenced lines. Furthermore, we demonstrated that NtBBX9 and NtBBX11 regulated the cold stress response by alternating the expression of critical cold-regulated genes such as CBFs, LEA14, and LTI65 and reactive oxygen species (ROS) production. The combination of NtBBX9 and NtBBX11 could facilitate tobacco’s rapid response to and recovery from transient cold stress and regulate plant cold-responsive growth in tropical regions.

As an effective antioxidant enzyme, superoxide dismutase (SOD) has been widely used as a food supplement, cosmetic additive, and therapeutic agent. However, oral delivery of SOD is challenging due to its relative instability, limited bioavailability, and low absorption efficiency in the gastrointestinal (GI) tract. We addressed these issues using a highly stable superoxide dismutase (hsSOD) generated from a hot spring microbial sample. This SOD exhibited a specific activity of 5000 IU/mg while retaining its enzymatic activity under low pH environments of an artificial GI system and in the presence of surfactants and various proteolytic enzymes. The inhibitory effects of hsSOD against skin-aging was evaluated under both in vitro and in vivo experiments using fibroblast cell and D-galactose induced aging-mouse models, respectively. Effective oral delivery of hsSOD promises wide applicability in pharmaceutical and food industries.

Abstract Thermoprofundales, formerly Marine Benthic Group D (MBG-D), is a ubiquitous archaeal lineage found in sedimentary environments worldwide. However, its taxonomic classification, metabolic pathways, and evolutionary history are largely unexplored because of its uncultivability and limited number of sequenced genomes. In this study, phylogenomic analysis and average amino acid identity values of a collection of 146 Thermoprofundales genomes revealed five Thermoprofundales subgroups (A–E) with distinct habitat preferences. Most of the microorganisms from Subgroups B and D were thermophiles inhabiting hydrothermal vents and hot spring sediments, whereas those from Subgroup E were adapted to surface environments where sunlight is available. H2 production may be featured in Thermoprofundales as evidenced by a gene cluster encoding the ancient membrane-bound hydrogenase (MBH) complex. Interestingly, a unique structure separating the MBH gene cluster into two modular units was observed exclusively in the genomes of Subgroup E, which included a peripheral arm encoding the [NiFe] hydrogenase domain and a membrane arm encoding the Na+/H+ antiporter domain. These two modular structures were confirmed to function independently by detecting the H2-evolving activity in vitro and salt tolerance to 0.2 M NaCl in vivo, respectively. The peripheral arm of Subgroup E resembles the proposed common ancestral respiratory complex of modern respiratory systems, which plays a key role in the early evolution of life. In addition, molecular dating analysis revealed that Thermoprofundales is an early emerging archaeal lineage among the extant MBH-containing microorganisms, indicating new insights into the evolution of this ubiquitous archaea lineage.

Background Dandruff is a chronic, recurring, and common scalp problem that is caused by several etiopathogeneses with complex mechanisms. Management of this condition is typically achieved via antifungal therapies. However, the precise roles played by microbiota in the development of the condition have not been elucidated. Despite their omnipresence on human scalp little is known about the co-occurrence/co-exclusion network of cutaneous microbiota. Results We characterized the scalp and hair surface bacterial and fungal communities of 95 dandruff-afflicted and healthy individuals residing in China. The degree distributions of co-occurrence/co-exclusion network in fungi-bacteria and bacteria-bacteria were higher in the healthy group ( P < 0.0001), whereas the betweenness values are higher in the dandruff group ( P < 0.01). Meanwhile, the co-occurrence/co-exclusion network among fungi-fungi and fungi-bacteria showed that compared to the healthy group, the dandruff group had more positive links ( P < 0.0001). In addition, we observed that Malassezia slooffiae , Malassezia japonica and Malassezia furfur , were more abundant in the dandruff group than in the healthy group. These microbiota were co-exclusion by either multiple bacterial genera or Malassezia sp. in healthy group. The lactic acid bacteria on the scalp and hair surface, especially the genera Lactobacillus and Lactococcus , exhibit a negative correlation with multiple bacterial genera on the scalp and hair surface. Lactobacillus plantarum and Pediococcus lactis isolated on the healthy human scalp can inhibit the growth of Staphylococcus epidermidis in vitro. Conclusions We showed that microbial networks on scalp and hair surface with dandruff were less integrated than their healthy counterparts, with lower node degree and more positive and stronger links which were deemed to be unstable and may be more susceptible to environmental fluctuations. Lactobacillus bacteria have extensive interactions with other bacteria or fungi in the scalp and hair surface micro-ecological network and can be used as targets for improving scalp health.

将木质纤维素转化为可发酵糖用于生产生物燃料以及生物基化学品是实现碳中和的有效途径之一.木质纤维素降解酶在这一过程中发挥着重要作用.里氏木霉是应用最为广泛的纤维素酶、半纤维素酶工业生产菌株.长期以来,里氏木霉一直被认为是红褐肉座菌的无性型,只能进行无性繁殖,菌种改良以经典诱变、基因育种等为主.直到近些年才证实里氏木霉可以进行有性生殖,这一发现为里氏木霉通过遗传有性杂交育种提供了可能性.本文对里氏木霉有性生殖的分子机制以及研究进展进行了综述,并结合作者多年来在里氏木霉菌种改造中的工作积累,对里氏木霉菌株的遗传改良新策略提出展望.

The genetic and metabolic diversity of deep-sea microorganisms play important roles in phosphorus and sulfur cycles in the ocean, distinguishing them from terrestrial counterparts. Malathion is a representative organophosphorus component in herbicides, pesticides, and insecticides and is analogues of neurotoxic agent. Malathion has been one of the best-selling generic organophosphate insecticides from 1980 to 2012. Most of the sprayed malathion has migrated by surface runoff to ocean sinks, and it is highly toxic to aquatic organisms. Hitherto, there is no report on bacterial cultures capable of degrading malathion isolated from deep-sea sediment. In this study, eight bacterial strains, isolated from sediments from deep-sea hydrothermal regions, were identified as malathion degradators. Two of the tested strains, Pseudidiomarina homiensis strain FG2 and Pseudidiomarina sp. strain CB1, can completely degrade an initial concentration of 500 mg/L malathion within 36 h. Since the two strains have abundant carboxylesterases (CEs) genes, malathion monocarboxylic acid (MMC α and MMC β) and dibasic carboxylic acid were detected as key intermediate metabolites of malathion degradation, and the pathway of malathion degradation between the two strains was identified as a passage from malathion monocarboxylic acid to malathion dicarboxylic acid.

Background Ergothioneine (ERG) is a potent histidine-derived antioxidant that confers health-promoting effects. Only certain bacteria and fungi can biosynthesize ERG, but the ERG productivity in natural producers is low. ERG overproduction through genetic engineering represents an efficient and cost-effective manufacturing strategy. Results Here, we showed that Trichoderma reesei can synthesize ERG during conidiogenesis and hyphal growth. Co-expression of two ERG biosynthesis genes ( tregt 1 and tregt 2) from T. reesei enabled E. coli to generate 70.59 mg/L ERG at the shaking flask level after 48 h of whole-cell biocatalysis, whereas minor amounts of ERG were synthesized by the recombinant E. coli strain bearing only the tregt 1 gene. By fed-batch fermentation, the extracellular ERG production reached 4.34 g/L after 143 h of cultivation in a 2-L jar fermenter, which is the highest level of ERG production reported thus far. Similarly, ERG synthesis also occurred in the E. coli strain engineered with the two well-characterized genes from N. crassa and the ERG productivity was up to 4.22 g/L after 143 h of cultivation under the above-mentioned conditions. Conclusions Our results showed that the overproduction of ERG in E. coli could be achieved through two-enzymatic steps, demonstrating high efficiency of the fungal ERG biosynthetic pathway. Meanwhile, this work offers a more promising approach for the industrial production of ERG.

Organophosphorus compounds (OPCs), including highly toxic nerve agents and pesticides, have been used widely in agricultural and military applications. However, they have aroused widespread concern because they persistently pollute the environment and threaten human life. Organophosphorus acid anhydrolase (OPAA) is a promising enzyme that can detoxify OPCs. Here, a novel OPAA (OPAA114644) was isolated and characterized from deep-sea sediment (−3104 m). It exhibited excellent alkaline stability, and the loss of activity was less than 20% in the pH range 5.0–9.0, even after being incubated for 30 d at 4 °C. It also exhibited high salt tolerance, and its enzymatic activity increased by approximately fourfold in the presence of 20% NaCl (w/v). Additionally, OPAA114644 exhibited high degradation efficiency for soman, dichlorvos, paraoxon, coumaphos, and chlorpyrifos with a concentration of up to 250 mg/L, with the degradation rate being 100%, 100%, 100%, 80% and 51%, respectively, in 20 min under optimal conditions. Notably, OPAA114644 dissolved in different solutions, such as 20% NaCl, 1 mM SDS, 0.05% soap, 10% methanol, and tap water, could efficiently decontaminate the residual paraoxon on the surfaces of glasses, cotton tissues, and apples. These results indicate that OPAA114644 has excellent potential for the biodegradation and bioremediation of OPCs pollution and represents a real application of OPAA in the decontamination and detoxification of foods and clothes, and in the remediation of sites such as floors. Deep-sea sediment might also be an abundant resource for various functional microorganisms and enzymes.

Enzymes that can decompose synthetic plastics such as polyethylene terephthalate (PET) are urgently needed. Still, a bottleneck remains due to a lack of techniques for detecting and sorting environmental microorganisms with vast diversity and abundance. Here, we developed a fluorescence-activated droplet sorting (FADS) pipeline for high-throughput screening of PET-degrading microorganisms or enzymes (PETases). The pipeline comprises three steps: generation and incubation of droplets encapsulating single cells, picoinjection of fluorescein dibenzoate (FDBz) as the fluorogenic probe, and screening of droplets to obtain PET-degrading cells. We characterized critical factors associated with this method, including specificity and sensitivity for discriminating PETase from other enzymes. We then optimized its performance and compatibility with environmental samples. The system was used to screen a wastewater sample from a PET textile mill. We successfully obtained PETdegrading species from nine different genera. Moreover, two putative PETases from isolates Kineococcus endophyticus Un-5 and Staphylococcus epidermidis Un-C2-8 were genetically derived, heterologously expressed, and preliminarily validated for PET-degrading activities. We speculate that the FADS pipeline can be widely adopted to discover new plastic-degrading microorganisms and enzymes in various environments and may be utilized in the directed evolution of degrading enzymes using synthetic biology.

A novel artificial Zinc finger -luciferase fusion protein was successfully developed for rapid detection of Salmonella typhimurium, a worldwide-distributed foodborne pathogen. The designed Zinc finger (ZF) protein bound specifically to a 12 bp region of the Salmonella spp invasion gene invA. While the luciferase from Gaussia princeps called Gaussia luciferase (Gluc) was for the first time fused with the artificial ZF domain to improve the detection sensitivity. The fusion protein successfully recognized and bound to the synthesized invA dsDNA with high specificity and sensitivity. The detection limit was as low as 10 fmol of dsNDA. Then, the bacteria PCR products were subsequently used to assess the zinc finger -luciferase fusion protein. The final results indicated that the ZF-Gluc fusion protein system could detect S. typhimurium as low as 1 CFU/mL in 2 h after the PCR. Therefore, this study provided us with a novel artificial zinc finger fusion protein and an efficient method to accomplish the rapid detection of the major foodborne pathogen S. typhimurium. In addition, the specific artificial ZF proteins that bund to particular dsDNA sequences could be easily designed, the ZF-Gluc might has broad application prospects in the field of rapid pathogenic bacteria detection. (c) 2021 Elsevier Inc. All rights reserved.

Vib1, a member of the Ndt80/PhoG-like transcription factor family, has been shown to be essential for cellulase production of Trichoderma reesei. Here, we combined transcriptomic and genetic analyses to gain mechanistic insights into the roles of Vib1 during cellulose degradation. Our transcriptome analysis showed that the vib1 deletion caused 586 genes with decreased expression and 431 genes with increased expression on cellulose. The downregulated genes were enriched for Gene Ontology terms associated with carbohydrate metabolism, transmembrane transport, oxidoreductase activity, and transcription factor activity. Of the 258 genes induced by cellulose, 229 showed no or decreased expression in Δvib1 on cellulose, including almost all (hemi)cellulase genes, crucial sugar transporter genes (IDs:69957, 3405), and the genes encoding main transcriptional activators Xyr1 and Ace3. Additionally, Vib1 also regulated the expression of genes involved in secondary metabolism. Further comparison of the transcriptomes of Δvib1 and Δxyr1 in cellulose revealed that the genes regulated by Vib1 had much overlap with Xyr1 targets especially for the gene set induced by cellulose, presumably whose expression requires the cooperativity between Vib1 and Xyr1. Genetic evidence indicated that Vib1 regulates cellulase gene expression partially via Xyr1. Our results will provide new clues for strain improvement.

[目的]建立适用于海洋微生物的流式细胞分选与高通量单细胞培养的方法,通过该方法从印度洋深海样品中分离微生物纯培养菌株.[方法]利用流式细胞仪单细胞分选功能,以前向角(FSC)和侧向角(SSC)散射光信号代替荧光信号作为分选逻辑,对深海水体和沉积物样品中微生物进行单细胞高通量分选和培养.[结果]确定了流式细胞分选的区域和条件,发现所建立方法适于分离海洋水体微生物,而不是沉积物微生物.从印度洋深海水体样品中获得61个潜在新菌株,分属于6个新属种,占分离菌株总数的26.29％,其16S rRNA基因序列与已培养的模式菌株相似性为89.79％-95.37％.[结论]本研究所建立的方法有助于提高发现海洋微生物新物种的效率,获得更多新的海洋微生物资源.

极端环境微生物定义了生命的边界.为了适应各种极端环境,极端环境微生物通过合成许多独特的活性化合物来保护自己.四氢嘧啶就是其中一种代表性的保护性物质.它最早是从极端嗜盐菌中发现的,作为调节细胞渗透压的一类相容性溶质,可以帮助微生物适应高盐等恶劣环境.研究发现四氢嘧啶不仅是一种重要的渗透压调节剂,还是一种高效的生物保护剂,可以帮助蛋白、核酸、生物膜乃至整个细胞对抗高温、干燥、冷冻和辐射等多种逆境.因此,四氢嘧啶在生物保护、生物医药和生物科技等众多领域展现出广阔的商业化应用前景.随着合成生物学和代谢工程技术的快速发展,传统的嗜盐菌四氢嘧啶生产方法已逐步被产率更高、环境更友好的生物工程菌及技术所取代.本文围绕四氢嘧啶的微生物合成及其应用研究进行综述,为后续四氢嘧啶的开发和应用提供重要参考.