title: Investigation on the biosynthesis of polyketide products in Aspergillus ustus and cyclodipeptide derivatives in Streptomyces strains
title: Untersuchung zur Biosynthese von Polyketid-Produkten in Aspergillus ustus und Cyclodipeptid-Derivaten in Streptomyces-Stämmen
titleType: AlternativeTitle
Creators: name: Yang, Yiling
Publisher: name: Philipps-Universität Marburg
Year: 2023
Subjects: Biosynthese
Polyketid
Pharmacology &amp; therapeutics, prescription drugs
Pharmakologie, Therapeutik
Streptomyces-Stämme
Aspergillus ustus
Streptomyces strains
biosynthesis
Cyclodipeptid-Derivate
cyclodipeptide derivatives
polyketide

description: Natural products have high structural diversity with various pharmacological or biological activities, which are of great significance to our life and drug research. Millions of natural products with versatile structural diversity have been found in nature. In recent years, a large number of microbial genome sequences have been released in public databases and revealed many silent or cryptic secondary metabolite gene clusters hidden in their genomes. This shows the great potential for discovering new metabolites. Advances in sequencing technology and bioinformatics analysis also provide great advantages for studying the biosynthesis and structural diversity of these metabolites. Structural differentiation of natural products begins with the formation of basic scaffolds using basic building blocks derived from primary metabolism catalyzed by different backbone enzymes. The structural complexity of natural products mainly arises from tailoring enzymes to highly functionalize the skeletons with a set of chemical transformations. The well-studied modification enzymes range from different types of oxidoreductases, cytochrome P450 enzymes, to various prenyltransferases (PTs) and methyltransferases (MTs). In addition, nonenzymatic events have also contributed to the formation of final products with vast diversity and complexity. Therefore, fully exploring these unexplored gene clusters and the substrate promiscuity of enzymatic and non-enzymatic reactions for the natural product formation may be a promising way and a new strategy to explore the metabolite diversity. In a cooperation study with Dr. Liujuan Zheng, the biosynthesis of a highly oxygenated phenethyl derivative ustethylin A, isolated from Aspergillus ustus, was elucidated. Due to the instability of ustethylin A, it was acetylated before isolation and structure elucidation. Gene deletion and heterologous expression proved that the phenethyl core structure is assembled by a polyketide synthase (UttA) harboring a methyltransferase domain. Isotopic labelling experiments proved that the backbone of ustethylin A is derived from malonyl-CoA and the methyl groups, also in the phenethyl residue, are from L-methionine. Modifications on the core structure by an aryl acid reductase (UttJ), a putative nonheme FeII/2-oxoglutarate dependent oxygenase (UttH), a cytochrome P450 enzyme (UttC) and a O-methyltransferase (UttF) led to the final product ustethylin A. This study is the first report on the biosynthetic pathway of a phenethyl-containing natural product. In cooperation with Dr. Jing Liu, the biosynthesis of streptoazine C and guanitrypmycin D1 was elucidated. Firstly, a three-gene cluster coding for a cyclodipeptide synthase, a prenyltransferase, and a methyltransferase was identified in Streptomyces aurantiacus by genome mining. Heterologous expression and precursor incubation experiments led to the elucidation of the biosynthetic steps of streptoazine C. In vivo biotransformation experiments proved the high flexibility of the prenyltransferase SasB toward tryptophan-containing cyclodipeptides and their dehydroderivatives for regular C-3-prenylation. This study provides an enzyme with a high substrate promiscuity from the less explored prenyltransferase group in cyclodipeptide synthase-related pathways. Afterwards, a two-gene cluster coding for a CDPS and a cytochrome P450 was identified in Streptomyces sp. NRRL S-1521 by phylogenetic analysis. Heterologous expression and structural elucidation of the isolated products proved that the cytochrome P450 GutD1521 catalyzes the regiospecific transfer of guanine to C-2 of the indole ring of cyclo-(L-Trp-L-Tyr) via a C−C linkage, which represents a new chemical transformation within this enzyme class. Precursor incubation experiments revealed that GutD1521 efficiently accepts several other tryptophan-containing cyclodipeptides or derivatives for regiospecific coupling with guanine, thus generating different guanitrypmycin analogs. This study provides a biocatalyst for a new linkage pattern between a indole ring and a guanine moiety and expands the functional spectrum of P450s as tailoring enzymes.