Biosynthesis of penilactones and peniphenones in Penicillium crustosum

Secondary metabolites originated from plants, bacteria and fungi constitute a large group of compounds, which are not essential for the growth, development and reproduction of the organism, but necessary for protection, competition and species interactions. Microbes, e.g. fungi and bacteria, have be...

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Bibliographic Details
Main Author: Fan, Jie
Contributors: Li, Shu-Ming (Prof. Dr.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2020
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Summary:Secondary metabolites originated from plants, bacteria and fungi constitute a large group of compounds, which are not essential for the growth, development and reproduction of the organism, but necessary for protection, competition and species interactions. Microbes, e.g. fungi and bacteria, have been more important sources of natural products since the discovery of penicillin in 1928. With advanced isolation and characterization techniques for secondary metabolites from crude biological samples, diverse compounds from different groups including polyketides, nonribosomal peptides, alkaloids and terpenes have been identified. Producing organisms utilize a limited set of primary metabolic building blocks to produce different natural product skeletons, which are further modified by a number of tailoring enzymes to form a variety of end products. For example, a core structure of polyketide can be derived from acyl-CoAs by polyketide synthase(s) (PKS(s)) and catalyzed by a series of tailoring enzymes such as nonheme FeII/2-OG-dependent oxygenases, flavin-containing oxidoreductases, cytochrome P450s and prenyltransferases to create an amazing diversity of natural product architectures. To facilitate the biosynthetic mechanism, advanced bioinformatics, biological technologies and biochemical tools have been utilized to investigate the coding genes of these enzymes, which are usually located together as a biosynthetic gene cluster (BGC). However, post-biosynthetic non-enzymatic events can also be involved in natural product formation. In this thesis, biosynthesis of secondary metabolites from a fungal strain, Penicillium crustosum PRB-2, was investigated in cooperation with Ge Liao. Penilactones A, B and D, as well as peniphenone D, structurally comprising clavatol and r-butyrolactone moieties, were identified from the wild type. Two separate gene clusters were functionally characterized as building blocks of the complex penilactone and peniphenone structures by gene disruption in the native PRB-2 strain, heterologous expression in Aspergillus nidulans and precursor feeding experiment in the available deletion mutants. A non-reducing (NR) PKS ClaF from the clavatol cluster is responsible for the formation of clavatol. A hybrid PKS-NRPS TraA from the terrestric acid cluster is involved in the biosynthesis of crustosic acid and terrestric acid, which undergo C-C bond cleavage to give r-butyrolactone moieties in penilactones and peniphenones. Oxidation of clavatol to hydroxyclavatol by a nonheme FeII/2-OG-dependent oxygenase ClaD and its spontaneous dehydration to an intermediate ortho-quinone methide initiate the non-enzymatic 1,4-Michael additions with r-butyrolactones. Therefore, the cross-coupling of two moieties from two separate gene clusters leads to the formation of peniphenone D and penilactone D, which undergo a second Michael addition with ortho-quinone methide to give penilactones A and B. Our findings represent rare examples of complex structures derived from two separate clusters and formed through enzymatic and non-enzymatic approaches. Afterwards, the investigation on terrestric acid formation was extended by using similar strategies. The hybrid PKS-NRPS TraA and the enoyl reductase TraG were demonstrated to be responsible for the accumulation of the tetronate core structure carboxylcrustic acid and viridicatic acid as precursors of crustosic acid in PRB-2. Biochemical characterizations proved that the conversion of crustosic acid to terrestric acid was achieved via oxidative decarboxylation catalyzed by a nonheme FeII/2-OG-dependent oxygenase TraH and subsequent stereospecific C-C double bond reduction by a flavin-containing oxidoreductase TraD. Among the two-step oxidative decarboxylation and stereospecific reduction, the mechanism with FeIV=O species as important intermediates was postulated for TraH-catalyzed olefination with or without CO2 elimination. Results on the biosynthesis of terrestric acid also provide a valid experimental basis for understanding the formation of the fungal acyltetronates with different stereochemistry involving sequential redox-assisted decarboxylation and stereoisomerization. In addition to penilactones and peniphenones, there are more clavatol-containing natural products from fungi. We wondered that these compounds are very likely synthesized from different precursors by nucleophilic attacking ortho-quinone methide derived from hydroxyclavatol. This hypothesis triggered our interest to screen the reactivity of ortho-quinone methide with diverse natural products or natural product-like compounds. Coincubation of 102 selected reactants with hydroxyclavatol under mild conditions (in nearly pH neutral aqueous solution) led to the detection of clavatol coupling products in 86 cases. As a result, 32 new clavatol-containing compounds were identified after isolation and structural elucidation. The conjugation between clavatol and the nucleophiles occurs mainly with the C-C bond formation at para- or ortho-positions of hydroxyl/amino group at the benzene ring and C-2 position of the indole skeleton. This study confirmed the activity of the ortho-quinone methide which is spontaneously derived from hydroxyclavatol in an aqueous system and increased significantly the diversity of clavatol-containing products in nature.
Physical Description:293 Pages