Background: Terrein, a major secondary metabolite from Aspergillus terreus, shows great potentials in biomedical and agricultural applications. However, the low fermentation yield of terrein in wild A. terreus strains limits its industrial applications. Results: Here, we constructed a cell factory based on the marine-derived A. terreus RA2905, allowing for overproducing terrein by using starch as the sole carbon source. Firstly, the pathway-specific transcription factor TerR was over-expressed under the control of a constitutive gpdA promoter of A. nidulans, resulting in 5 to 16 folds up-regulation in terR transcripts compared to WT. As expected, the titer of terrein was improved in the two tested terR OE mutants when compared to WT. Secondly, the global regulator gene stuA, which was demonstrated to suppress the terrein synthesis in our analysis, was deleted, leading to greatly enhanced production of terrein. In addition, LS-MS/MS analysis showed that deletion of StuA cause decreased synthesis of the major byproduct butyrolactones. To achieve an optimal strain, we further refactored the genetic circuit by combining deletion of stuA and overexpression of terR, a higher terrein yield was achieved with a lower background of byproducts in double mutants. In addition, it was also found that loss of StuA (both ΔstuA and ΔstuA::OEterR) resulted in aconidial morphologies, but a slightly faster growth rate than that of WT. Conclusion: Our results demonstrated that refactoring both global and pathway-specific transcription factors (StuA and TerR) provides a high-efficient strategy to enhance terrein production, which could be adopted for large-scale production of terrein or other secondary metabolites in marine-derived filamentous fungi.

Harmful algal blooms (HABs), mainly formed by dinoflagellates, have detrimental effects on marine ecosystems and public health. Therefore, detecting HABs is crucial for early warning and prevention of HABs as well as the mitigation of their adverse effects. Although various methods, such as light microscopy, electron microscopy, real-time PCR, and microarrays, have already been established for the detection of HABs, they are still cumbersome to be exploited in the field. Therefore, rapid nucleic detection methods such as recombinase polymerase amplification (RPA) and loop-mediated isothermal amplification (LAMP)-lateral flow dipstick (LFD) have been developed for monitoring bloom-forming algae. However, the CRISPR/Cas-based detection of HABs has yet to be applied to this field. In this study, we developed a method for detecting Karenia mikimotoi (K. mikimotoi), a typical ichthyotoxic dinoflagellate responsible for global blooms. Our method utilized Cas12a from Lachnospiraceae bacterium ND2006 (LbCas12a) to target and cleave the internal transcribed spacer (ITS) of K. mikimotoi, guided by RNA. We leveraged the target-activated non-specific single-stranded deoxyribonuclease cleavage activity of LbCas12a to generate signals that can be detected using fluorescence-read machines or LFDs. By combining RPA and LbCas12a with reporters, we significantly enhanced the sensitivity, enabling the detection of ITS-harboring plasmids at concentrations as low as 9.8 aM and genomic DNA of K. mikimotoi at levels as low as 3.6 × 10⁻⁵ ng/μl. Moreover, we simplified the genomic DNA extraction method using cellulose filter paper (CFP) by directly eluting the DNA into RPA reactions, reducing the extraction time to < 30 s. The entire process, from genomic DNA extraction to result reporting, takes less than an hour, enabling the identification of nearly a single cell. In conclusion, our method provided an easy, specific, and sensitive approach for detecting K. mikimotoi, offering the potential for efficient monitoring and management of K. mikimotoi blooms.

Histone acetylation modification significantly affects secondary metabolism in filamentous fungi. However, how histone acetylation regulates secondary metabolite synthesis in the lovastatin (a lipid-lowering drug) producing Aspergillus terreus remains unknown because protein is involved and has been identified in this species. Here, the fungal-specific histone deacetylase gene, hstD, was characterized through functional genomics in two marine-derived A. terreus strains, Mj106 and RA2905. The results showed that the ablation of HstD resulted in reduced mycelium growth, less conidiation, and decreased lovastatin biosynthesis but significantly increased terrein biosynthesis. However, unlike its homologs in yeast, HstD was not required for fungal responses to DNA damage agents, indicating that HstD likely plays a novel role in the DNA damage repair process in A. terreus. Furthermore, the loss of HstD resulted in a significant upregulation of H3K56 and H3K27 acetylation when compared to the wild type, suggesting that epigenetic functions of HstD, as a deacetylase, target H3K27 and H3K56. Additionally, a set of no-histone targets with potential roles in fungal growth, conidiation, and secondary metabolism were identified for the first time using acetylated proteomic analysis. In conclusion, we provide a comprehensive analysis of HstD for its targets in histone or non-histone and its roles in fungal growth and development, DNA damage response, and secondary metabolism in A. terreus.