title: Genomics-driven and biochemical approaches to expand the spectrum of natural products
title: Genomikgestützte und biochemische Ansätze zur Erweiterung des Spektrums natürlicher Produkte
titleType: AlternativeTitle
Creators: name: Li, Wen
Publisher: name: Philipps-Universität Marburg
Year: 2023
Subjects: Biosynthese
biosynthesis
Pharmacology &amp; therapeutics, prescription drugs
Pharmakologie, Therapeutik
natürliche Produkte
Penicillium
prenyltransferases
secondary metabolite
Prenyltransferasen
Sekundärmetabolite

description: Natural products (NPs) derived from secondary metabolism of living organisms play a pivotal role in drug discovery, especially for the treatment of cancer and infectious diseases. Their versatile skeletons are synthesized by different biosynthetic enzymes including polyketide synthases (PKSs), nonribosomal peptide synthetases (NRPSs), terpene cyclases, and hybrid enzymes. Further modifications by tailoring enzymes such as oxidoreductases, cytochrome P450 enzymes, and prenyltransferases (PTs) increase the structural diversity and improve their biological and pharmacological activities. With the advances in genome sequencing and analytical technologies, many strategies have been applied for exploring the promising categories for drug discovery. Due to low or no expression of the majority of biosynthetic gene clusters (BGCs) in the native genomes, it is of great significance to activate such promiscuous BGCs for new drug leads. In recent years, genome mining of novel BGCs has achieved significant progress in NP discovery. In addition to secondary metabolite (SM) exploration in microorganisms, the substrate-based enzymatic reactions have also been proven to be a useful tool for enriching the chemical database. The members of dimethylallyl tryptophan synthase (DMATS) superfamily as important biocatalysts were widely used for structural modification of diverse small molecules. Vast of new prenylated structures have been obtained through chemoenzymatic synthesis. In this thesis, we identified an α-pyrone derivative by genome mining of a NRPS-PKS gene in Penicillium crustosum and obtained a series of prenylated cyclodipeptide (CDP) analogs through chemoenzymatic synthesis of different DMATSs. In the first project, a novel NRPS-PKS hybrid gene pcr10109 from Penicillium crustosum PRB-2 was chosen for detailed investigation by Dr. Jie Fan. She cloned the gene into the expression vector for heterologous expression in Aspergillus nidulans. Analysis of the SMs and structure elucidation proved its responsibility for 4-hydroxy-6-(4-hydroxyphenyl)-2H-pyran-2-one production. Further isotopic feeding experiments revealed its biosynthetic pathway. Para-hydroxybenzoic acid (PHBA) as the precursor and two acetate molecules are assembled for final product formation. To increase the product yield, we fed PHBA in the cultures and the product yield reached a maximum of 51 mg/kg rice culture, which is five-fold higher than that obtained without feeding. This provides another method to increase product formation by supplementing of special substrates. In the second project, we mainly focused on the production of diprenylated cyclo-L-Trp-L-Pro (cWP). At first, we intended to follow the nature´s biosynthetic machinery by utilizing C2-PT EchPT1 as the first biocatalyst. However, the C2-prenylated cWP could not be accepted by C4-, C5-, C6-, and C7-PTs for further prenylation. Dr. Lindsay Coby found that the C2-PT EchPT1 can also catalyze prenylations of monoprenylated cyclodipeptides. Then we changed the strategy and firstly obtained the C4-, C5-, C6-, C7-monoprenylated cWP in high product yields. After that, the monoprenylated derivatives were incubated with EchPT1 for the reverse C2-prenylation. Large scale enzyme assays and NMR analysis proved the products to be C2,C4-, C2,C5-, N1,C6-, and C2,C7-diprenylated cWP. This is the first report that EchPT1 can also catalyze the prenylation at the N1 position of the indole ring. In the third project, a similar method was used for the production of prenylated tryptophan-containing dimeric CDPs. We chose different dimeric CDPs and PTs for the enzyme activity test. cyclo-L-Trp-L-Trp (cWW) dimers tetratryptomycins A − C were well accepted by EchPT1 in the presence of DMAPP. Tetratryptomycins A and C are better substrates of EchPT1 for prenylation compared with tetratryptomycin B. Compound isolation and NMR analysis determined the products as C2- (and C2´-) prenylated tetratryptomycins, which is consistent with EchPT1-catalyzed reactions. Further kinetic parameter determination revealed that the values are in the range of EchPT1-catalyzed reactions toward most CDPs.