title: Investigation on the biosynthesis of polyketides in two Penicillium strains
title: Untersuchung zur Biosynthese von Polyketiden in zwei Penicillium-Stämmen
titleType: AlternativeTitle
Creators: name: Xiang, Pan
Publisher: name: Philipps-Universität Marburg
Year: 2022
Subjects: Pharmacology &amp; therapeutics, prescription drugs
Pharmakologie, Therapeutik
Penicillium
polyketides
biosynthesis

description: Secondary metabolites, a special class of natural products, generally display significant biological activities in organisms, especially those from plants, bacteria, and fungi. For example, diverse natural products with remarkable structures have been found in fungi. Most of these compounds can be further categorized into four main classes based on their biosynthetic origins: polyketides, peptides, alkaloids, and terpenoids. Polyketide natural products have revolutionized medicine and drastically improved our lives by providing such important active substances as drugs to fight cancer, pathogens, and autoimmune diseases. Advances in sequencing technologies and bioinformatic analysis have facilitated the elucidation of the biosynthesis of these natural products. Their structural divergence begins with the formation of the initial skeletons by different backbone enzymes using fundamental metabolic building blocks derived from primary metabolism. For example, the carbon backbone for polyketides is derived from acyl-CoA and constructed by the core enzymes polyketide synthases (PKSs), which belong to one of the most studied enzyme classes in the last decades. The fungal aromatic polyketides are mainly synthesized by nonreducing polyketide synthases (NRPKSs), in which no reduction is employed during the elongation of the polyketide chain. Subsequent modifications of the backbone structures are catalyzed by tailoring enzymes, such as transferases and oxidoreductases, to form diverse and complex pathway products. In this thesis, two NRPKS genes were identified in Penicillium crustosum and functionally confirmed by heterologous expression, domain deletion and recombination, as well as by feeding experiments. Expression of the NRPKS gene pcr9304 in the established and frequently used host Aspergillus nidulans led to the accumulation of three isocoumarins, proving its function as an isocoumarin synthase. Precursor feeding experiments revealed that the endogenous enzymes from A. nidulans can modify the initial PKS product by hydroxylation and methylation. These results provided one additional example that unexpected further modifications can take place in a heterologous host. Heterologous expression of another NRPKS gene, oesA from P. crustosum, led to the identification of 3-orsellinoxypropanoic acid. Domain deletion and recombination demonstrated that OesA with a domain structure of SAT-KS-AT-PT-ACP1-ACP2-TE catalyzed not only the formation of orsellinic acid, but also its transfer to 3-hydroxypropanoic acid, proving its role as a bifunctional enzyme, i.e. orsellinic acid synthase and transferase. Both ACP domains contribute independently and complementarily to the product formation. Isotopic labelling experiments proved that only the orsellinyl residue of 3-orsellinoxypropanoic acid is derived from acetate. In cooperation with Bastian Kemmerich, the biosynthetic pathway of annullatins in Penicillium roqueforti was elucidated. An eleven-gene anu cluster was identified in P. roqueforti by genome mining. The involvement of the anu cluster in the biosynthesis of annullatin D with a fused dihydrobenzofuran lactone ring system and its derivatives was confirmed by heterologous expression of the whole cluster in A. nidulans. A combinational approach of in vitro enzymatic studies and heterologous expression was used to understand the formation of the five-member lactone ring in annullatin D. The aromatic backbone is assembled by a cooperation of the highly reducing polyketide synthase AnuA, together with two additional enzymes AnuBC. Then, the polyketide core structure is consecutively modified by hydroxylation at the C5 alkyl chain with the cytochrome P450 AnuE. The prenyltransferase AnuH subsequently installs one isoprenyl group at the benzene ring. Afterward, enzymatic or non-enzymatic dihydrobenzofuran ring formation between the prenyl and the phenolic hydroxyl groups in the prenylated product results in two diastereomers. Among them, the (2S, 9S)-configured isomer is converted to annullatin D by the BBE-like enzyme AnuG for the five-member lactone ring formation. The (2R, 9S)-isomer is likely very instable and immediately oxidized by the short-chain dehydrogenase/reductase AnuF to annullatin F. This study demonstrated a highly programmed and efficient biosynthetic pathway for annullatins. Despite the intriguing structural features and biological activities, biosynthetic studies on annullatins, especially on the formation of the lactone ring in annullatin D have not been reported prior to our study. Furthermore, we identified a new BBE-like enzyme for oxidative lactonization between two hydroxyl groups.