Investigation of biotechnologically relevant enzymes ofnatural product biosynthesis in microorganisms
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Philipps-Universität Marburg
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Abstract
Microorganisms are omnipresent on our planet. They produce natural products whose
derivatives make up a considerable proportion of the drugs in use today. Natural products are
divided into primary metabolites, which are essential for survival, and secondary metabolites
(SM), which are not essential but represent a selective advantage for the producer. Secondary
metabolites are found in a large number of living organisms and have a wide variety of
biological and pharmacological effects. The biosynthesis of SM begins mainly through the
formation of a backbone structure. This structure allows various modifications by so-called
modifying enzymes. The modifying enzymes in SM-associated biosynthesis pathways include
prenyltransferases (PTs) and cytochrome P450 enzymes (P450s).
The initial section of the thesis examines these PTs. PTs have proven to be invaluable tools in
the synthesis of new chemical structures and drug candidates. These enzymes are widely
distributed in nature and participate in a multitude of biosynthetic pathways. While numerous
dimethylallyl tryptophan synthase-PTs from ascomycetes have been identified and
characterized, surprisingly little is known about these enzymes from basidiomycetes. At the
time of this study, only two members of this class had been described. The prenyltransferase
ShPT from the basidiomycete Stereum hirsutum exhibited a striking degree of similarity to a
previously described PT involved in the biosynthesis of vibralactone. It was therefore
postulated that ShPT might also prenylate 4-hydroxybenzyl alcohol. We received a codon-
optimized plasmid containing the PT from our cooperation partners at the Interfaculty Institute
of Biochemistry at the University of Tübingen, for the heterologous expression in E. coli. In the
initial phase of this project, I conducted a genomic analysis of ShPT and overexpressed the PT
in E. coli. A biochemical analysis of this PT was conducted to ascertain its acceptability of
different substrates. The results demonstrate that benzyl alcohol derivatives are not accepted
as substrates for enzymatic catalysis by ShPT. Instead, hydroxynaphthalenes are identified as
the preferred major prenyl acceptors. Consequently, ShPT is the first basidiomycete
prenyltransferase to be characterized as accepting hydroxynaphthalenes as substrates.
Furthermore, the results illustrate that ShPT exhibits a broad substrate specificity towards
DMAPP and GPP as prenyl donors. Subsequently, further studies were conducted on the
neighboring cytochrome P450 enzyme by heterologously expressing it in Aspergillus nidulans
LO8030 and subsequently culturing the transformant in the presence of prenylated
hydroxynaphthalene. While the natural substrate of ShPT remains unidentified, our findings
indicate that ShPT catalysis expands the chemical arsenal of enzymes from the DMATS
superfamily, enabling the production of new regioselectively prenylated naphthalenes. This
provides a new approach for the future discovery and synthesis of new drug candidates. The second part of this work deals with P450 enzymes. Six orthologous two-gene BGCs for
mycocyclosin and guatyromycine biosynthesis were identified by our research group.
Biochemical studies have confirmed that the P450 enzymes GymBx act as intramolecular
oxidases and intermolecular nucleobase transferases. These enzymes facilitate oxidative C-C
coupling in cYY, or alternatively, link it to guanine or hypoxanthine. This represents a distinctive
attribute of CDP-modifying P450 enzymes. In collaboration with the University of Tübingen,
we undertook an investigation into the structure of this unusual enzyme family and sought to
elucidate the mechanism of its chemoselective catalysis. Jasmin Freytag and Dr. Georg Zocher
were able to solve the crystal structures of two members, GymB1 and GymB5, in their unbound
state, as well as that of GymB5 in complex with hypoxanthine and cYY. The structural data of
the ternary complex facilitated an understanding of CDP binding and the nucleobase transfer
reaction. This enables the identification of crucial positions for substrate recognition and
modification of the bifunctional enzymes. By side-directed and saturation mutagenesis
performed by me, a GymB5 variant was created that exclusively exhibits intramolecular
coupling enzyme activity. These data provide a robust basis for future protein modification of
P450 enzymes to synthesize new CDP nucleobase adducts and other "unnatural" natural
products.
Through genome mining, we identified several interesting cytochrome P450 enzymes that may
be involved in tailoring reactions in the third part of this thesis. Through heterologous
expression in S. albus J1074 I elucidated the function of a CDPS backbone gene and
demonstrated the involvement of a P450 enzyme in hydroxylation reactions. Precursor feeding
experiments led to the generation of the same new products in the native host strain.
Furthermore, an attempt was made to elucidate the function of another previously
uncharacterized actinomycete BGC by heterologous expression in S. coelicolor M1146. The
resulting accumulated product was isolated and the structure elucidated.
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Dates
Created: 2024Issued: 2025-11-26Updated: 2024-11-04
Faculty
Fachbereich Pharmazie
Language
eng
Data types
DoctoralThesis
Keywords
protein engineeringgenome miningprenyltransferasegenetic engineeringcytochrome p450
DFG-subjects
Stereum hirsutumStreptomycesNaturstoffBiosynthese
DDC-Numbers
570
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Martin, Andreas: Investigation of biotechnologically relevant enzymes ofnatural product biosynthesis in microorganisms. : Philipps-Universität Marburg 2025-11-26. DOI: https://doi.org/10.17192/z2024.0479.