1 1.7 Future Perspectives
1.8 Scope of This Thesis
Natural product discovery started with penicillin in 1928 and has been facilitated in recent years by microbial genomes sequencing programs. A number of these natural products found their application as immunosuppressive-, cytostatic-, or cholesterol-reducing agents. In this thesis, the filamentous fungus Penicillium chrysogenum, known for its β-lactam production, was investigated. The industrial derivatives of P. chryso-genum strain have found further applications as cell factories for the biosynthesis of complex peptides.
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1.8. Scope of This Thesis 27
The aim of this thesis was to uncover the unexploited source of novel secondary metabolites with biological activity in industrial strains of P. chrysogenum. The ma-jority of them are derived from either Nonribosomal Peptide Synthetase (NRPS), Polyketides synthetase (PKS), or Polyketide Synthetase-Nonribosomal Peptide Syn-thetase (PKS-NRPS) hybrids. However, not all the NRPS clusters are expressed un-der standard growth conditions in relevant strains of P. chrysogenum. The activation and elucidation of these silent secondary metabolites gene clusters were performed followed by analysis of the corresponding products formation.
The current understanding of fungal secondary metabolites biosynthesis path-ways and many aspects of their transcriptional regulation are presented in Chapter 1. This overview focuses on strategies applied in searching novel bioactive com-pounds through targeted activation of silent gene clusters in filamentous fungi. Po-tential drug discovery may be achieved by direct engineering the secondary metabo-lite gene cluster or by simulation of physiological conditions in the species’ natural ecosystem. Alternatively, this can be achieved by applying more a general approach such a manipulation of global regulators including enzymes involved in mediating the change in chromatin structure.
Chapter 2presents the role of the global secondary metabolite regulator LaeA on transcriptional profile and secondary metabolites production in industrial strains of P. chrysogenum. Overexpression of LaeA did not alter the penicillin production but did influence the transcription and compound’s formation in other biosynthetic pathways. The effect of LaeA-like methyltransferases (Llm) on industrial strains of P. chrysogenum is shown in Chapter 3. The deletion of two highly expressed PcLlm7 and PcLlm3 encoding genes resulted in transcriptional activation of silent pks11. The impact on pks11 was strongly enhanced in double deletion mutant suggesting that both PcLlm proteins act as negative regulators of secondary metabolism in P. chryso-genum industrial strains.
Chapter 4 describes secondary metabolites controlled by histone deacetylase (HdaA) in P. chrysogenum industrial strains. The hdaA deletion mutant produced new metabolites and showed the activation of a silent dual PKS containing pathway in which pks12 and pks13 are transcribed in opposite direction. At the same time, green conidial pigmentation and chrysogine biosynthesis were decreased.
Cultivation of P. chrysogenum under iron limited conditions induced the expres-sion of three silent nrps identified as siderophore synthetases (Pss), as character-ized in Chapter 5. Synthesis of ferrichrome, coprogen, dimerum acid, N-acetyl-fusarinine, fusarinine A and numerous new siderophores were assigned to the re-spective Pss genes. Severe growth impairment was observed for the ∆pssA and
∆pssB mutants in iron depletion solid media, although not for the ferrichrome syn-thetase mutant (∆pssC), suggesting distinct physiological roles for the siderophores produced by PssC on the one hand, and PssA and PssB on the other hand. Addi-tionally in Chapter 6, four genes involved in siderophore transport were targeted
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for inactivation.Chapter 7describes approach based on genetic introduction of the strong PpcbC promoter in front of silent and low-expressed NRPS and HPN encoding genes in industrial strains of P. chrysogenum. This resulted in transcriptional activation and subsequent metabolites formation. These metabolites were abundant in the media of overexpression strains; they were not detectable, or present only in very low quantity in the wild type, and completely absent in the deletion strains.
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