Universal Silent Aim
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These examples demonstrate that there is no universal price ceiling actually existing. And since, as Henderson concedes, price rises in one field affect all prices, the cost of living will continue to rise while wages remain frozen. And that is inflation.
Secondary metabolites (SMs) are biologically active organic compounds that are biosynthesised by many plants and microbes. Many SMs that affect the growth, behaviour or survival of other organsisms have been re-purposed for use as medicinal drugs, agricultural biocides and animal growth promoters. The majority of our anti-infective and anti-cancer drugs are currently derived from Streptomyces, bacteria that are free living, filamentous, and ubiquitous in terrestrial habitats. Genome sequencing and mature in silico approaches to genome mining has revealed that filamentous fungi contain very large numbers of genes related to SM production. Yet these genes are typically silent under laboratory conditions. There are now many tools and strategies available to activate or clone silent SM genes. This thesis details our efforts to apply various methods to define and then manipulate SM genes in Cladosporium fulvum, a biotrophic pathogenic fungus of tomato containing many silent SM genes and gene clusters.
In chapter 1, the relevance of SMs to medicine and agriculture is considered. Filamentous fungi are presented as untapped sources of potential useful SMs, as their genomes are often rich in SM biosynthetic genes that are silent under most conditions. Methods to activate these silent genes and increase the chemical diversity of fungi are detailed. These include the deletion or over-expression of genes encoding regulatory proteins, the use of chemical inhibitors, and the manipulation of growth conditions. Heterologous expression of silent SM genes in a production host is also discussed as a tool for bypassing host regulatory mechanisms altogether. C. fulvum is introduced as an organism that has been intensively studied as a biotrophic plant pathogen. Genomic analysis showed that this fungus has twenty-three core SM genes, a large catalogue composed of 10 polyketide synthases (PKSs), 10 non-ribosomal peptide synthases (NPS), one PKS-NPS hybrid and one dimethylallyl tryptophan synthase (DMATS). Transcriptional profiling showed that the majority was silent during growth on tomato and in vitro. Cladofulvin is introduced as the sole detectable SM produced by C. fulvum during growth in vitro. This presented an opportunity to apply the aforementioned strategies to induce these silent genes and obtain new compounds. The importance of cladofulvin and structurally related anthraquinones are briefly discussed as potential medicines. The value of the cladofulvin biosynthetic gene cluster is also emphasised as a potential source of novel biosynthetic enzymes.
In chapter 2 the SM gene catalogue identified during the analysis of the C. fulvum genome was analysed in further detail. Each locus containing a core SM gene was inspected for other biosynthetic genes linked to SM production, such as those encoding decorating enzymes and regulators. Products of these SM genes or gene clusters were speculated, based on their similarity to those characterized in other fungi. Six gene clusters were located in the genome of C. fulvum that are conserved in other fungal species. Remarkably, two predicted functional gene clusters were linked to the production of elsinochrome (PKS1) and cercosporin (PKS7), toxic perylenequinones that generate reactive oxygen species (ROS). We profiled the expression of core SM genes during the growth of C. fulvum under several in vitro conditions. Expression of each core SM gene was measured by RT-qrtPCR and the resulting SM profile was determined by LC-MS and NMR analyses. Confirming previous findings, the majority of SM genes remained silent and only cladofulvin was detected. During growth on tomato only two core genes, PKS6 and NPS9, were clearly expressed, but both were significantly down-regulated during colonization of the mesophyll tissue of tomato leaves. We confirmed that cladofulvin does not cause necrosis on solanaceous plants when infiltrated into their leaves. In contrast to other biotrophic fungi that have a reduced SM production capacity, our studies of C. fulvum suggest that down-regulation of SM biosynthetic pathways might represent another mechanism associated with a biotrophic lifestyle.
In chapter 3 our efforts to activate cryptic pathways in C. fulvum are described, with the aim of discovering new compounds. Many Ascomycete-specific global regulators of SM production and morphological development in other fungi were identified in C. fulvum. We investigated three intensively studied regulators, VeA, LaeA and HdaA. Deleting or over-expressing the genes encoding these regulators in C. fulvum yielded no new detectable SMs. Cladofulvin biosynthesis was strongly affected by each regulator; HdaA is an activator while VeA and LaeA are repressors of cladofulvin production. Attempts were made to stimulate SM production in the mutants and wild type strains by growing them on different carbon sources, but only cladofulvin biosynthesis was affected. Interestingly, cladofulvin production was stimulated by carbon limitation and strongly repressed in the presence of saccharose. Similar to observations made in other fungi, the deletion of VeA or LaeA did not affect viability, but maturation and conidiation were affected. Sporulation was not overtly affected by the loss of HdaA, but Δhdaa deletion mutants did not produce cladofulvin. This suggests that cladofulvin production is not required for asexual reproduction. The main finding of this chapter is that global regulator manipulation cannot considered to be a universal tool to discover new fungal natural products. 59ce067264
