High-copy integration of key genes offers a promising strategy for efficient biosynthesis of valuable natural products in Saccharomyces cerevisiae. However, traditional multi-copy gene integration methods meet challenges including low efficiency and labor-intensive screening processes. In this study, we developed the IMIGE (Iterative Multi-copy Integration by Gene Editing) system, a CRISPR/Cas9-based approach that exploits both δ and rDNA repetitive sequences for simultaneous multi-copy integrations in S. cerevisiae. This system combines the mixture of Cas9-sgRNA expression vectors with a split-marker strategy for efficient donor DNA assembly in vivo and enables rapid, iterative screening through growth-related phenotypes. When applied to the biosynthesis of ergothioneine and cordycepin, the IMIGE system achieved significant yield improvements, with titers of 105.31 ± 1.53 mg/L and 62.01 ± 2.4 mg/L, respectively, within just two screening cycles (5.5–6 days in total). These yields represent increases of 407.39 % and 222.13 %, respectively, compared to the strains with episomal expression. By streamlining the integration process, utilizing growth-based selection, and minimizing screening demands in both equipment and labor, the IMIGE system could provide an efficient and scalable platform for high-throughput strain engineering, facilitating enhanced microbial production of a wide range of bioproducts.

Cordycepin, an adenosine analog, exhibits diverse bioactivities and holds significant potential for applications in healthcare and agriculture. Fungi of the genus Cordyceps, such as Cordyceps militaris, can naturally produce cordycepin. Current sources of cordycepin primarily involve extraction from fruiting bodies or isolation from liquid fermentation using C. militaris, presenting challenges such as low production intensity, complex separation and purification systems, and high production costs, limiting industrial feasibility. Recent advancements have witnessed the utilization of various fungal chassis cells to successfully engineer heterologous biosynthetic platforms for cordycepin, such as Saccharomyces cerevisiae and unconventional yeasts, offering advantages of high yield, short fermentation cycles, and a broad substrate spectrum. This mini review summarizes the biosynthetic pathways of cordycepin and focused on the comparison of the characteristics, advantages, current performance and prospects for the microbial cell factories, analyzing potential targets for metabolic pathway modification and giving strategies in both genetic engineering and process engineering to enhance production intensity. The mini review particularly emphasizes the crucial role of chassis cell stress tolerance to the toxic product in determining cordycepin yield and highlights the urgent need for high-throughput screening methods for high-yield strains.

Priestia megaterium (P. megaterium PH3) is an endophytic bacterium isolated from peanuts. It has natural resveratrol production ability and shows potential application value. This study analyzed its genetic function and metabolic mechanism through whole-genome sequencing and found that the genome size is 5,960,365 bp, the GC content is 37.62%, and 6132 genes are annotated. Functional analysis showed that this strain contained 149 carbohydrate active enzyme genes, 7 secondary metabolite synthesis gene clusters, 509 virulence genes, and 273 drug-resistance genes. At the same time, this strain has the ability to regulate salt stress, low temperature, and hypoxia. Genomic analysis reveals a stilbene-synthase-containing type III polyketide synthase gene cluster that contributes to resveratrol synthesis. A safety assessment showed that the strain is non-hemolytic, does not produce amino acid decarboxylase, and is not resistant to multiple antibiotics. In the mouse model, P. megaterium PH3 did not have significant effects on body weight, behavior, or physiological indicators. These results provide important basic data and theoretical support for its industrial application and the research and development of plant protection agents.

Cordycepin, a nucleoside analog naturally produced by Cordyceps fungi, exhibits broad-spectrum biological activity and has been reported to inhibit both prokaryotic and eukaryotic microorganisms, although its antifungal mechanisms remain poorly understood. In this study, we systematically evaluated the inhibitory effects of cordycepin on the growth, development, and pathogenicity of Magnaporthe oryzae, the causal agent of rice blast, and further elucidated its mode of action through transcriptomic analysis and gene function validation. Cordycepin significantly inhibited hyphal growth and reduced both conidial production and germination in a dose-dependent manner. Moreover, it severely impaired appressorium formation and increased the frequency of malformed structures, ultimately leading to attenuated virulence. Transcriptome profiling revealed that cordycepin reprogrammed multiple metabolic and signaling pathways, including nitrogen metabolism, fatty acid metabolism, melanin and chitin biosynthesis, and protein kinase networks. Notably, two carbonic anhydrase genes, MoCA1 and MoCA5, were significantly downregulated and identified as candidate genes responsive to cordycepin. Functional analysis showed that MoCA5 localizes to mitochondria and interacts with MoCA1; deletion of either gene resulted in disrupted mitochondrial membrane potential, reduced ATP synthesis, and decreased pathogenicity. Collectively, our results indicate that cordycepin suppresses the growth and virulence of M. oryzae through coordinated modulation of mitochondrial function and nitrogen metabolism - associated pathways. These findings provide new insights into the antifungal action of cordycepin and identify potential molecular components involved in fungal metabolic adaptation to cordycepin stress.