title: Investigation on the biosynthesis of nitrogen-containing metabolites in different microorganisms
title: Untersuchungen zur Biosynthese stickstoffhaltiger Metabolite in verschiedenen MikroorganismenLösungsmittel Wasser.
titleType: AlternativeTitle
Creators: name: Dai, Yu
Publisher: name: Philipps-Universität Marburg
Year: 2024
Subjects: secondary metabolites
PKS-NRPS-Hybrid
Biosynthese
PKS-NRPS hybrid
Pharmacology &amp; therapeutics, prescription drugs
Pharmakologie, Therapeutik
Cyclodipeptid
Streptomyces
biosynthesis,
cyclodipeptide
Penicillium
Indole diterpene
Sekundärmetaboliten
Indol-Diterpen

description: Natural products (NPs), typically considered as secondary metabolites (SMs), are not essential for an organism’s growth or reproduction and development, but provide significant adaptive advantages. These compounds, which enhance the competitiveness and survival of organisms in their environments, have long been recognized as a treasure trove of bioactive molecules. Their extensive use spans across human and veterinary medicine, as well as agriculture. In fact, approximately half of the drugs approved by the FDA in the past 40 years are related to NPs, highlighting their crucial role in drug discovery and development.
Microbial sources, particularly fungi and bacteria, have been key to NP discovery since the breakthrough with penicillin in 1928. Advances in biological and analytical techniques have significantly expanded our ability to explore and harness the vast biochemical potential of microorganisms. Additionally, the study of NP biosynthesis offers insight into generating new bioactive compounds. Among these, nitrogen-containing natural products are especially prominent due to their prevalence in pharmaceuticals, functional materials, and biologically active molecules. In this thesis, the focus is placed on the biosynthesis of various nitrogen-containing metabolites, which hold potential for the discovery of new bioactive compounds.
In the first project, we demonstrated the formation of an eight-membered C−N hetero ring via nonenzymatic flavin-conducted oxidative indole ring opening. Five such indole diterpenes were converted to their 6/8/6/6/6 pentacyclic 2,18-dioxo-2,18-seco derivatives. Differing from usual flavin-related oxygenations by O2 activation with FMNH2, FMN in its oxidized form initiates the reactions by taking two electrons from the indole nitrogen or a hydroxyl group. More interestingly, isotopic-labeled experiments with 18O2 or H218O demonstrated that the incorporated oxygen atoms are originated from water instead of molecular O2. In this process, the oxidized form of flavin catalyzes two successive oxidations of amines to imines with involvement of hydrolysis for the ring expansion. The reduced flavin is then regenerated by oxidation with molecular oxygen to form H2O2, which was proved by H2O2 detection and quantitative assay.
In the second project, which was carried out in cooperation with Daniel Ostendorff, we proved the function of a three-gene cluster from Streptomyces albofaciens, coding for a cyclodipeptide synthase (CDPS) SalbA and two cytochrome P450 enzymes SalbB and SalbC. SalbA was identified as a cWL synthase by expression in Escherichia coli and production isolation. Heterologous expression of genes in various combinations in Streptomyces coelicolor and cultivation of the transformants with and without substrates revealed that SalbB and SalbC utilized cWL for divergent metabolism, i.e. cWL guaninylation by SalbB and cWL hydroxylation by SalbC. The pathway products were isolated and their structures were elucidated by extensive spectroscopic analysis. In addition, cultivation in medium containing 15NH4Cl provides evidence for the incorporation of a guanine moiety in the guaninylated product guatrypleumycine A. X-ray crystallographic study confirmed the stereospecific hydroxylation at C-10 of the tryptophanyl residue in the hydroxylated product cyclo(trans-10-hydroxy-L-Trp-L-Leu). Moreover, SalbC is the first β-hydroxylase for cyclodipeptide identified in a CDPS-associated biosynthetic pathway.
In the third project, which was cooperated with Dr. Wen Li, we identified a PKS-NRPS hybrid contained biosynthetic gene cluster pem in Penicillium crustosum and its products penilactams A – C, three enantiomeric pairs of tetramate derivatives. Overexpression of the positive regulator pemC significantly improved the product yields. Gene deletion and feeding experiments with 15N-glycin proved that the tetramic acid units are products of glycine with one or two C2 units co-catalyzed by a PKS-NRPS hybrid enzyme and a trans-acting enol reductase. Feeding with hydroxyclavatol provided evidence for the formation of penilactams A – C via nonenzymatic 1,4-Michael addition of tetramic acids with the highly reactive ortho-quinone methide.