[Objective] To explore the technical feasibility of using transgenic microalgae to control Aedes that transmit diseases such as dengue fever. [Methods] Taking the neurotransmitter transporter gamma-aminobutyric acid receptor gene (gat) of Aedes as a target, we constructed a shRNA-expressing vector and then transferred the vector into Chlamydomonas reinhardtiii CC124 and Chlorella vulgaris HOC5 through electroporation. The recombinant microalgae were used to feed the larvae and adults of Aedes albopictus. [Results] The transgenic algae delayed the larval development and had obvious lethal effects on the larvae and adults of A. albopictus, causing the mortality rates of 75.56% and 58.67%, respectively. The expression level of gat in the larvae was significantly down-regulated. [Conclusion] Deploying gat-shRNA transgenic microalgae in enclosed water environments to suppress mosquito populations in the vicinity is technically feasible. This strategy provides a new perspective on using biological methods to control mosquitoes and block the transmission of severe infectious diseases such as dengue fever and Zika virus disease.

[Objective] To investigate the inhibitory effect and mechanism of thymol on the biofilm formation of Proteusmirabilis (PM). [Methods] The minimal inhibitory concentration (MIC) and minimal biofilm inhibitory concentration (MBIC) of thymol were determined by the broth micro-dilution method. The time-kill curve of thymol was established by the colony counting method. Crystal violet staining was performed to determine the inhibitory effect of thymol on the biofilm formation of PM. The effects of thymol on the swarming and swimming of PM and the extracellular polymeric substances (extracellular polysaccharides, extracellular proteins, and eDNA) of the biofilm were determined. RT-qPCR was employed to investigate the effects of thymol on the expression levels of PM biofilm-related genes: rsbA (encoding histidine-containing phosphotransmitter of the bacterial two-component system), flhD (encoding the flagellar regulon), and mrpA (encoding mannose-resistant Proteus-like fimbriae). [Results] The MIC of thymol against PM was 0.156 mg/mL. Thymol significantly inhibited the biofilm formation of PM, with the MBIC of 0.313 mg/mL. Thymol inhibited the motility of PM, reduced the formation of extracellular polysaccharides, proteins, and eDNA in PM biofilm, and decreased the expression levels of rsbA, flhD, and mrpA. [Conclusion] Thymol inhibits the biofilm formation of PM by suppressing the motility and formation of extracellular polymers and down-regulating the expression of biofilm-related genes.

[Objective] Streptomyces rapamycinicus has large biosynthetic potential with 55 natural product biosynthetic gene clusters (BGCs), most of which have not yet been identified. This study aims to obtain a series of novel compounds by Bacterial Artificial Chromosome (BAC) library-based cloning of novel BGCs from S. rapamycinicus on a large scale and then heterologously expressing them in model Streptomyces strains. [Methods] The bioinformatics analysis of the novelty of BGCs screened out 11 unknown BGCs encoding non-ribosomal peptides, polyketides or terpenoids, from S. rapamycinicus SIPI RP202. Then, we cloned these BGCs by constructing a BAC library and screening via PCR, and then introduced them into three heterologous expression hosts by conjugative transfer. Finally, LC-MS was employed to detect whether these BGCs were successfully expressed after fermentation in three media and fermentation broth extraction with two approaches. Novel compounds were separated, purified, and structurally elucidated. [Results] An unknown terpenoid BGC was successfully expressed in Streptomyces albus Del14. Three novel aromatic meroterpenoids, rapamylic acids A-C, were identified. Then, the potential biosynthetic pathways of rapamylic acids A-C were proposed based on their structural features and BGC. [Conclusion] We successfully unlocked a silent BGC from S. rapamycinicus by large-scale BGC cloning and heterologous expression, providing an alternative strategy for the activation of silent BGCs from other Streptomyces strains. Meanwhile, the discovery of this kind of novel meroterpenoids expands the structural diversity of bacterial terpenoids.

Edible forest mushrooms are one of the main forest foods and an essential resource for forests to achieve the functions of conserving water, bringing in economic benefits, boosting grain production, and also serving as a carbon sink. This study summarizes the current situation of the edible forest mushroom industry in China, such as benign development with imbalanced regional development, scaled planting under forests but lack of regional characteristics and deep processing products, and the advantages of edible ectomycorrhizal mushrooms with weak scientific and technological support. Suggestions are put forward on optimizing planning, promoting the investment of scientific and technological resources, accelerating digital intelligence and multi-mode development, conducting climate change monitoring, and protecting germplasm resources, in order to make positive contributions to the development of the edible forest mushroom industry and provide scientific and technological support for the practice of the big food concept.

A mutant strain G₉-72 with a high yield of ectoine was obtained from wild type Halomonas campaniensis after nine rounds of ultraviolet mutagenesis. The differentially expressed genes/proteins (DEGs/DEPs) and the molecular mechanism underlying the excessive increase in the ectoine yield remain to be explored for the mutant strain. [Objective] To explore the DEGs/DEPs between the wild type strain XH26 and G₉-72 and decipher the molecular mechanism of efficient ectoine production by conjoint analysis. [Methods] A non-salt (NS, 0 mol/L NaCl) group and a high-salt (HS, 1.5 mol/L NaCl) group were designed for the culture of XH26 and G₉-72. Illumina HiSeq and quantitative mass spectrometry were employed to identify the DEGs/DEPs between the two strains by transcriptomics-proteomics conjoint analysis. Furthermore, RT-qPCR was carried out to verify the expression of significant DEGs. [Results] The transcriptomics analysis revealed 11 amino acid metabolic pathways (44 DEGs) associated with ectoine anabolism, and the proteomics analysis revealed ten amino acid metabolic pathways (50 DEPs) associated with ectoine anabolism. The transcriptomics-proteomics conjoint analysis identified 15 significant DEGs, including seven genes (ectB, betB, betA, asd, doeD, doeC, and gabD) with up-regulated mRNA and protein level, four genes (ItaE, gdhA, gabT, and acnB) with down-regulated mRNA and protein levels, three genes (gltD, atoB, and narG) with down-regulated mRNA levels and up-regulated protein levels, and one gene narK with up-regulated mRNA level and no protein level. Additionally, the RT-qPCR results were consistent with the transcriptomics analysis. [Conclusion] The excessive increase in the ectoine yield of the mutant strain was associated with key genes in the ectoine metabolic pathway (including the synthesis genes asd and ectB and the catabolism genes doeD and doeC) and indirectly associated with several genes (betB, betA, ItaE, gltD, gadA, and acnB) in the upstream metabolic pathway. Notably, ectoine biosynthesis was highly associated with the Ala/Asp/Glu/His metabolic pathway (gabD, gdhA, gabT, and atoB) and nitrogen source metabolism (narK and narG).

The dinitroaniline herbicide pendimethalin, as a pre-emergent herbicide, is widely employed for weed control in cotton fields across Xinjiang. Characterized by chemical stability, prolonged residual activity, bioaccumulative potential, and biomagnification, it is extensively applied in agricultural practice, leading to increased risks to soil ecosystems. Accordingly, the removal of pendimethalin residues has garnered increasing attention. [Objective] To enrich the microbial consortia with pendimethalin-degrading ability, study succession characteristics of microbial consortia during the enrichment culture process under pendimethalin stress, and identify the key microorganisms involved in pendimethalin degradation. [Methods] The cotton field soil under long-term pendimethalin stress was inoculated into MSM media with pendimethalin at 0, 1.2, and 12 mg/L, respectively. The succession of the microbial consortium structure under pendimethalin stress was investigated by high-throughput sequencing. [Results] Two microbial consortia capable of degrading pendimethalin were enriched. Among them, L4 (low-concentration group) achieved a degradation rate of 100% for 1.2 mg/L pendimethalin within 11 days, while H4 (high concentration group) showed a degradation rate of 37.2% for 12 mg/L pendimethalin over the same period. The alpha diversity of microbial consortia was considerably decreased by pendimethalin stress, and the bacteria responded to the stress more strongly than fungi. The microbial consortium structure varied with different concentrations of pendimethalin. The network stability, complexity, and modularity were diminished by pendimethalin stress. Linear discriminant analysis effect size (LEfSe) results showed that the specific bacterial taxa in the high concentration group were Achromobacter, Leifsonia, Candidatus_Nucleicultrix, Enterobacter, and Chryseobacterium. The specific bacterial taxa in the low concentration group were Methyloversatilis, Pseudoxanthomonas, Ancylobacter, Methylorubrum, Thermomonas, and Pseudoflavitalea. Talaromyces, Trichoderma, Paracremonium, Scedosporium, and Sarocladium were the specific fungal taxa. The PICRUSt2 analysis showed the pendimethalin stress significantly enriched the pathways related to degradation. The correlation analysis between microbial genera and pendimethalin degradation showed that Methylorubrum, Hyphomicrobium, Microbacterium, Rhodopseudomonas, and Fusarium had positive correlations with pendimethalin degradation in the low concentration group. Hyphomicrobium, Leifsonia, Rhodopseudomonas, Talaromyces, and Trichoderma were positively correlated with pendimethalin degradation in the high concentration group. [Conclusion] Two microbial consortia capable of degrading pendimethalin were successfully obtained through enrichment culture under varying concentrations of pendimethalin. Leveraging high-throughput sequencing, this study systematically explored the succession patterns of microbial consortia under pendimethalin stress. Key functional microorganisms associated with pendimethalin degradation were preliminarily identified. The findings provide a theoretical basis for the targeted screening of efficient microbial strains dedicated to pendimethalin degradation.

[Objective] Chitin lyases are the key enzymes for bacteria to degrade chitin. Pseudoalteromonas arabiensis N1230-9 possesses five chitin lyase-encoding genes (woc28159, woc28160, woc28161, woc27404, and woc27232). Functional identification of these five genes is crucial for determining the ability of strain to degrade chitin. [Methods] The distribution patterns of five chitin lyases from strain N1230-9 in 44 model strains of Pseudomonas were analyzed by bioinformatics methods. The transcription levels of five chitin lyase-encoding genes of strain N1230-9 cultured in the medium with chitin as the sole carbon source were analyzed by RT-qPCR. The mutants with deletion of chitin lyase-encoding genes were constructed by homologous recombination, and their abilities to degrade chitin were evaluated. [Results] Thirty-three Pseudoalteromonas strains possessed chitin lyase homologous proteins of strain N1230-9. Chitin significantly upregulated the transcription levels of woc28159, woc28160, woc28161, and woc27404, and the deletion of each of the four genes weakened the chitin-degradation ability. The transcription level of woc27232 was not affected by chitin, and the deletion of this gene did not affect the chitin-degradation ability of the strain. [Conclusion] WOC28159 and WOC28161 are necessary for the degradation of chitin by strain N1230-9. WOC28160 and WOC27404 endow strain N1230-9 with efficient chitin-degrading ability, while WOC27232 does not participate in the chitin degradation process.

Plant endophytes are non-pathogenic microbial groups residing inside or in the interstices of plant tissue. They constitute a pivotal component of the plant microecological environment. These organisms are distinguished by their remarkable biodiversity and wide distribution across diverse regions of the host plant. Plant endophytes exhibit high species diversity, host plant diversity, habitat diversity, and functional diversity. They can secrete hormones that regulate plant growth and enhance the nutrient absorption capacity of their hosts to promote plant growth. Additionally, endophytes can promote plant growth indirectly by enhancing the host plant resistance to abiotic and biotic stresses. Notably, these endophytes are capable of producing substantial secondary metabolites, which exhibit antimicrobial, antiviral, antioxidant, and other biological activities. This capacity offers considerable potential for the development of novel pharmaceuticals, the extraction of natural products, and the creation of biopesticides. Endophytes have a wide range of applications in agriculture, industry, and medicine. They can be used as biocontrol agents to enhance crop yields or used for the production of natural pigments and perfumes and the development of novel pharmaceuticals. However, the research on plant endophytes still faces many challenges in species identification and function verification, molecular mechanism analysis of endophyte-host interactions, practical application technology, and safety evaluation.

[Objective] To better control the pollution of nitrate and sulfide in mariculture tailwater and reduce the ecological risks caused by the discharge of the tailwater, this study screened a halotolerant desulfurizing and denitrifying bacterial strain and studied its growth characteristics. [Methods] A dilution coating-repeat dish sandwish culture method was used to isolate and screen halotolerant desulfurizing and denitrifying bacterial strains, which were identified by morphological observation and 16S rRNA gene sequence comparison. Based on single factor experiments, the key factors affecting desulfurizing and denitrifying effects, including carbon source, temperature, salinity, pH, and inoculation amount, were optimized, and the strain tolerance threshold to sulfide (S^2-) was explored. [Results] A strain Stutzerimonas stutzeri D1-2 was isolated from sulfur-based mixotrophic denitrifying sludge. This strain was able to simultaneously remove sulfide and nitrate from the environment with organic carbon sources. With sodium lactate as the optimal carbon source, strain D1-2 showed the best performance at an inoculation amount of 1.5%. The strain showed the removal rates of S₂O₃^2- and NO₃^--N both greater than 80% at 15-35 ℃, salinities of 10‰-50‰, and initial pH 6.0-8.0. It demonstrated significant tolerance at an initial S^2- concentration of 50 mg/L, with the removal rates of S^2- and NO₃^--N reaching 97.91% and 94.67%, respectively. [Conclusion] This study reports the heterotrophic sulfur-oxidizing capacity of S. stutzeri. The halotolerant strain S. stutzeri D1-2 capable of simultaneously desulfurizing and denitrifying has a potential application value in the control of sulfide and nitrate pollution in mariculture tailwater.

The fruiting bodies of fungi of genus Shiraia inhabiting bamboo have a medicinal use in traditional Chinese medicine. Hypocrellin A (HA), the main bioactive perylenequinone pigment from S. bambusicola fruiting bodies is a novel non-porphyrin photosensitizer with antitumor and antimicrobial properties. [Objective] To investigate the effects of Shiraia fruiting body-associated fungi on HA biosynthesis and develop a co-culture method for enhancing HA production. [Methods] Shiraia fruiting body-associated fungi were isolated and the strains influencing HA biosynthesis were screened by a plate confrontation assay. The effects of intracellular and extracellular metabolites of the strains on HA production were evaluated. A co-culture system for Shiraia sp. S9 and associated fungi was established and optimized for enhancing HA production. [Results] There were 34 fungal strains including 6 host Shiraia strains isolated from the fruiting bodies. Among them, Fusarium sp. SF12 and its extracellular polysaccharides significantly promoted HA biosynthesis. Fusarium sp. SF12 did not noticeably affect the growth of Shiraia sp. S9 but regulated HA synthesis by upregulating the transcription levels of key enzyme genes involved in HA biosynthesis. The total HA yield was enhanced to 209.46 mg/L on day 8 after adding spores (100 cell/mL) from Fusarium sp. SF12 to the Shiraia culture at the time point of 24 h, which was 1.93 times that of the control. [Conclusion] There are diverse fungi in Shiraia fruiting bodies. The co-culture of the associated fungus Fusarium sp. SF12 and the host Shiraia sp. S9 is a new technique to improve HA production.