The biosynthesis of secondary metabolites isolated from filamentous fungi and the regulation of secondary metabolism in filamentous fungi and streptomyces
1) Glutathione is a ubiquitous thiol in eukaryotic cells, and its high intracellular ratio of reduced form (GSH) to oxidized form (GSSG) is largely maintained by glutathione reductase (GR) using NADPH as electron donor. glrA, a glutathione reductase encoding gene, was found and cloned from Acremonium chrysogenum by searching its genomic sequence based on similarity. Its deduced protein exhibits high similarity to GRs of other eukaryotic organisms. Disruption of glrA resulted in lack of GR activity and accumulation of a high level of GSSG in A. chrysogenum. Overexpression of glrA dramatically enhanced GR activity and the ratio of GSH/GSSG in this fungus. The spore germination and hyphal growth of glrA disruption mutant was strongly reduced in chemical defined medium. Meanwhile, the mutant was more sensitive to hydrogen peroxide than the wild-type strain. We found that the glrA mutant recovered normal germination and growth by adding exogenous methionine (Met). Exogenous Met also enhanced the antioxidative ability of both the mutant and wild-type strain. GSH determination indicated that the total GSH and ratio of GSH/GSSG in the mutant or wild-type strain were significantly increased when addition of Met into the medium. The glrA mutant grew poorly and could not produce detectable cephalosporin in the fermentation medium without Met. However, its growth and cephalosporin production was restored with addition of exogenous Met. These results indicate that glrA is required for the normal growth and protection against oxidative damage in A. chrysogenum, and its absence can be complemented by exogenous Met.
2) The thioredoxin system including thioredoxin and thioredoxin reductase (TrxR) is used for oxidative stress defenses in fungi. Based on the genomic sequence, a thioredoxin reductase-encoding gene (ActrxR1) was isolated from Acremonium chrysogenum CGMCC3.3795. Like other TrxRs, AcTrxR1 contains FAD binding domain, Redox domain, and NADPH binding domain. Disruption of ActrxR
3) T-DNA inserted mutants of Acremonium chrysogenum were constructed by Agrobacterium tumefaciens mediated transformation (ATMT). One mutant 1223 which grew slowly was selected. TAIL-PCR and sequence analysis indicated that a putative septation protein encoding gene AcsepH was partially deleted in this mutant. AcsepH contains nine introns, and its deduced protein AcSEPH has a conserved serine/threonine protein kinase catalytic (S_TKc) domain at its N-terminal region. AcSEPH shows high similarity with septation H proteins from other filamentous fungi based on the phylogenetic analysis of S_TKc domains. In sporulation (LPE) medium, the conidia of AcsepH mutant was only about one-seventh of the wild-type, and more than 20% of conidia produced by the mutant contain multiple nuclei which were rare in the wild-type. During fermentation, the AcsepH disruption mutant grew slowly and its cephalosporin production was only about one quarter of the wild-type, and the transcription analysis showed that pcbC expression was delayed and the expressions of cefEF, cefD1 and cefD2 were significantly decreased. The vegetative hyphae of AcsepH mutant swelled abnormally and hardly formed the typical yeast-like cells. The amount of yeast-like cells was about one-tenth of the wild-type after fermentation for 5 days. Comparison of hyphal viabilities revealed that the cells of AcsepH mutant died easily than the wild-type at the late stage of fermentation. Fluorescent stains revealed that the absence of AcsepH in A. chrysogenum led to reduction of septation and formation of multinucleate cells. These data indicates that AcsepH is required for the normal cellular septation and differentiation of A. chrysogenum, and its absence may change the cellular physiological status and causes the decline in cephalosporin production.
4) Ribosome assembly cofactor RimP is one of the auxiliary proteins required for maturation of the 30S subunit in Escherichia coli. Although RimP in protein synthesis is important, its role in secondary metabolites biosynthesis has not been reported so far. The rimP homologue rimP-SC was identified and cloned from Streptomyces coelicolor. Disruption of rimP-SC led to enhanced production of actinorhodin and calciumdependent antibiotics by promoting the transcription of actII-ORF4 and cdaR. Further experiments demonstrated that MetK was one of the reasons for the increment of antibiotics production. In addition, rimP-SC disruption mutant could be used as a host to produce more peptidyl nucleoside antibiotics (polyoxin or nikkomycin) than the wild-type strain. Likewise, disruption of rimP-SV of Streptomyces venezuelae also significantly stimulated jadomycin production, suggesting that enhanced antibiotics production might be widespread in many other Streptomyces species. These results established an important relationship between ribosome assembly cofactor and secondary metabolites biosynthesis and provided an approach for yield improvement of secondary metabolites in Streptomyces.