Molecular Basis Of The Secondary Metabolism In Chaetomium Globosum

Chaetomium globosum is one of most common fungi in nature. It is best known for producing chaetoglobosins, which belong to cytochalasans and inhibit movement and proliferation of mammalian cells due to their ability to bind to actin filaments. However, the molecular basis of chaetoglobosin biosynthesis including the key genes and the signaling pathways is poorly understood’in this fungus. C. globosum also have the ability to decomposite the cellulose effectively, but the molecular basis of cellulase system has remained uninvestigated. In addition, C. globosum has been widely investigated as a method for biological control.In this study, we first use a polyketide synthase gene, pks-1, to establish the technique of RNA interference(RNAi) in C. globosum and then characterized that this gene is involved in the production of chaetoglobosin A(ChA). ChA is the major component of the secondary metabolites in C. globosum and consists of a polyketide backbone that is condensed from nine units of acetate/malonate. When pks-1was knocked down by RNAi, the production of ChA dramatically decreased. Knock-down mutants also displayed a pigment-deficient phenotype. These results suggest that both polyketides, melanin and chaetoglobosin, are likely to share common biosynthetic steps. Most importantly, we found that pks-1also plays a critical role in sporulation. The silenced mutants of pks-1lost the ability to produce spores. We propose that polyketides may modulate cellular development via an unidentified action. Our results suggest that C. globosum pks-1is unique because of its triple role in melanin formation, chaetoglobosin biosynthesis and sporulation. This work implies an interconnection of chaetoglobosin biosynthesis, secondary metabolism and fungal morphogenesis.G protein-cAMP-PKA signaling pathway has been studied in numerous fungi and is recognized as one of the most important signaling pathways regulating secondary metabolite production. To investigate whether this signaling pathway regulate chaetoglobosin biosynthesis, in this study, a G-protein a-subunit-encoding gene, gna-1(G-protein alpha-subunit1), which encodes a protein showing a high degree of identity to Group Ⅰ a-subunits of fungal heterotrimeric G-proteins, was knock down by RNAi. Dozens of gnal-knock down strains (pG) and gnal/pkaR-knock. down strains (pGP) were obtained. The gna-1knock down mutants displayed a pigment-deficient phenotype which was similar to pks-1knock-down mutants. In the meanwhile, the concentration of ChA in the gnal knock-down transformants all fell sharply. These results suggest that melanin production, sporulation and ChA biosynthesis are all regulated by the G protein-cAMP-PKA signaling pathway.C. globosum also have the ability to decomposite the cellulose effectively. This work explored the cellulose-degradation system and factors on cellulase gene expression in Chaetomium globosum NK102. In the sequenced genome of NK102, we identified10cellulase genes by sequence homology alignment. We employed a high-throughput sequencing technology, RNA Sequcing (RNA-Seq), to monitor the differential expression of the genes under different culture conditions. We observed that cellulase activity generally increased with the time of the fungal cultures. Transcription level of the genes encoding cellobiohydrolase, cellobiose dehydrogenase and endoglucanase (cbhl, cdh and egll) was higher than the others. Expression of the transcriptional repressors, ACE Ⅰ and CreA, decreased with the culture age, whereas expression of Hap2/3/5complex was upregulated. In different carbon sources, cellulase activity and their gene transcription were repressed by glucose and were activated by cellobiose. While, sorbitol had no significant effect. Interestingly, light had a positive effect on the expression of these cellulase genes. This study detects the molecular system of cellulose-degradation in C. globosum and provides information for interpretation of carbohydrate metabolism.We also found that G protein-cAMP-PKA signaling pathway regulates cellulase gene expression in C. globosum NK102. RNA-Seq results showed that the expression of most carbon metabolism genes were down-regulated and the qRT-PCR result demonstrated cbh1、cbh2、egl1and egl2expression in gnal knock-down strains (pG) decreased sharply. The gnal/pkaR double knock down strains (pGP) or the activator of the cAMP pathway can restore the cellulose gene expression. In addition, the phenotype of the fungus grown on the cellulose medium and the cellulase activity results confirmed that cellulose degradation are regulated by the Gprotein-cAMP-PKA signaling pathway.The nematicidal activity of C. globosum NK102, culture filtrates and chaetoglobosin A (ChA) purified by HPLC were evaluated on Meloidogyne incognita. The result showed that C. globosum NK102significantly repelled second-stage juveniles (J2). Both filtrates and ChA demonstrated strong adverse effects on J2mortality of90.2%at300μg ChA/mL (LC50=126.5μg/mL). ChA and filtrates did not affect egg hatch until72h exposure. All filtrate treatments inhibited the J2penetration even in12.5%dilution treatment. Similarly, ChA (300and30μg/mL) showed a significant inhibitory effect on J2penetration. The number of egg per plant was significantly reduced by63%in the treatment of30mg ChA per kg soil relative to control plants, indicating an apparent negative effect on reproduction of M. incognita. The report suggests a potential for C. globosum as biocontrol agent for the integrated management of M. incognita.