Molecular biological and biochemical approaches to expand the spectrum of fungal natural products

At least 3.5 billion years ago, the first life on earth arose. This was the starting point of the evolutionary development of numerous living beings. According to current estimations, there are 1012 different species on our planet. Most of this enormous biodiversity originates from the kingdom of...

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Bibliographic Details
Main Author: Kindinger, Florian
Contributors: Li, Shu-Ming (Prof. Dr. ) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2019
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Summary:At least 3.5 billion years ago, the first life on earth arose. This was the starting point of the evolutionary development of numerous living beings. According to current estimations, there are 1012 different species on our planet. Most of this enormous biodiversity originates from the kingdom of bacteria and archaea. Based on these estimations, only 0.001 % of all species are known to this day. The omnipresent competition between living beings led to the development of secondary metabolism. The metabolites derived from this metabolism are not essential for survival, yet their production offers the organism various selection advantages. Plants, bacteria, and fungi are the main producers of secondary metabolites. The more than 2,140,000 million known secondary metabolites can be divided into five large groups: (1) non-ribosomal polypeptides, (2) polyketides, (3) alkaloids, (4) terpenoids and steroids and (5) enzyme cofactors. Many of these natural compounds show a biological or pharmaceutical activity and were used for the development of drugs. The large number of not yet identified microorganisms harbors an enormous, mostly unused genetic potential to produce further new natural compounds. Such compounds may be suitable for the development of urgently needed new drugs. Various approaches, such as heterologous expression in suitable host organisms, are being investigated to make this potential accessible. Additionally, through synthetic biology approaches, the diversity of natural substances can be further extended, and new natural substances can be discovered or produced. In the context of research on secondary metabolites, this work focuses on three main topics: 1. The extension of the spectrum of possible substrates for prenyltransferases, by using a database to predict new substrates. 2. The identification and characterization of previously unknown biosynthetic gene clusters, as well as the investigation of a possible application of the enzymes involved to produce new natural substances. 3. The generation of a host for the heterologous expression of secondary metabolite genes and investigation of their unknown products. Prenyltransferases catalyze the transfer of prenyl units (n × C5) to their target substrates. This is of importance, as an increase in the biological activity of prenylated compounds compared to their unprenylated counterparts has been observed for many compounds. A special property of prenyltransferases is their promiscuity with respect to the substrates. This makes them suitable candidates to produce pharmaceutically active substances. However, in practice, it is difficult to identify new substrates for prenyltransferases. In order to address this problem, a database, PrenDB, was developed for the prediction of such substrates. The predictive power of this database was experimentally tested with 38 predicted substrates by their acceptance with the prenyltransferases FtmPT1, FgapT2, and CdpNPT. For 27 of the 38 substrates, prenylation by at least one of the three tested enzymes was observed, 17 with conversion yields of more than 50 %. This proved the predictive power of the developed database and enabled the targeted selection of new potential substrates and the identification of new substrate classes. The identification of biosynthetic gene clusters and the subsequent biochemical characterization of the enzymes involved in the biosynthetic pathways form the basis for synthetic biology approaches to produce natural products. Based on the cyclic dipeptide echinulin, a possible procedure for the identification of the responsible gene cluster and the use of the involved enzymes for the biosynthesis of new substances was described. The enzymatic prerequisites for the biosynthesis of echinulin were determined based on the structural peculiarities of echinulin. Potential candidate gene clusters must encode one non-ribosomal peptide synthetase and several prenyltransferases. In the genome of the echinulin producer Aspergillus ruber, a gene cluster with these prerequisites was identified. Enzyme assays with the echinulin precursor cyclo-L-tryptophanyl-L-alaninyl and the heterologously produced prenyltransferases EchPT1 and EchPT2 led to a well-founded biosynthetic hypothesis and confirmed the involvement of this cluster in the biosynthesis of echinulin. The combination of EchPT1 and EchPT2 with cyclo-L-tryptophanyl-L-alaninyl as a substrate led to the formation of 7 products with different degrees of prenylation. This special property was subsequently used to prenylate further cyclic dipeptides. The stereoisomers of cyclo-tryptophanyl-alaninyl and cyclo-tryptophanyl-prolinyl were used for this purpose. Analogous to the biosynthesis of echinulin, this led to the formation of triprenylated main products prenylated at position C2, C5 and C7, as well as further di-, tri- and tetraprenylated side products. Another possibility to investigate and produce secondary metabolites is the heterologous expression in a suitable host. A potential new host for heterologous expression, Penicillium crustosum, was examined in this thesis. The genome of the fungus was sequenced and the involvement of the polyketide synthase Pcr4401 in the biosynthesis of the melanin precursor YWA1 was confirmed by deletion and expression experiments. Successful integration of foreign genes in the pcr4401 gene locus can easily be recognized by the occurrence of an albino phenotype. For better use as an expression host, a pyrG deficient strain and two plasmids were generated to integrate foreign genes into the pcr4401 gene locus. The applicability as an expression host was subsequently verified by the successful expression of three PKS genes and the structural elucidation of the formed products.
Physical Description:247 Pages