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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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| Format: | Doctoral Thesis |
| Language: | English |
| Published: |
Philipps-Universität Marburg
2019
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| Online Access: | PDF Full Text |
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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. |
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| Physical Description: | 247 Pages |
| DOI: | 10.17192/z2019.0516 |