Isolation and Purification of Natural Products from Microbial Cultures.

Antibiotic natural products from microbes are characterized by diverse and mostly complex chemical structures, which challenge their total chemical synthesis and make biotechnological production to the predominant production route. In order to reach these valuable compounds in the fermentation broth, sophisticated recovery methods are required, and a high degree of purity is essential for a thorough exploration of their beneficial properties in subsequent assays. The isolation and purification of natural products from microbial cultures is mainly based on the repeated application of extraction and chromatographic separation methods.This chapter describes the general strategy of natural product recovery from microbial cultures, gives theoretical and practical insights to underlying methods-essentially compound extraction and preparative chromatography-and describes a specific methodical approach to isolate and purify the natural product fusarubin from the culture of the fungus Fusarium sp.

Genome Assembly and Genetic Traits of the Pleuromutilin-Producer Clitopilus passeckerianus DSM1602.

The gilled mushroom Clitopilus passeckerianus (Entolomataceae, Agaricales, Basidiomycota) is well known to produce the terpenoid pleuromutilin, which is the biotechnological basis for medically important antibiotics such as lefamulin and retapamulin. Their unique mode of action and good tolerance entails an increasing demand of pleuromutilin-derived antibiotics in veterinary and human health care. Surprisingly, despite their pharmaceutical importance, no genome sequence is available of any pleuromutilin-producing fungus. Here, we present the high-quality draft genome sequence of the pleuromutilin-producer C. passeckerianus DSM1602 including functional genome annotation. More precisely, we employed a hybrid assembly strategy combining Illumina sequencing and Nanopore sequencing to assemble the mitochondrial genome as well as the nuclear genome. In accordance with the dikaryotic state of the fungus, the nuclear genome has a diploid character. Interestingly, the mitochondrial genome appears duplicated. Bioinformatic analysis revealed a versatile secondary metabolism with an emphasis on terpenoid biosynthetic enzymes in C. passeckerianus and also in related strains. Two alleles of biosynthetic gene clusters for pleuromutilin were found in the genome of C. passeckerianus. The pleuromutilin genes were reassembled with yeast-specific elements for heterologous expression in Saccharomyces cerevisiae. Our work lays the foundation for metabolic strain engineering towards higher yields of the valuable compound pleuromutilin.

The Endophytic Fungus Cyanodermella asteris Influences Growth of the Nonnatural Host Plant Arabidopsis thaliana.

Cyanodermella asteris is a fungal endophyte from Aster tataricus, a perennial plant from the northern part of Asia. Here, we demonstrated an interaction of C. asteris with Arabidopsis thaliana, Chinese cabbage, rapeseed, tomato, maize, or sunflower resulting in different phenotypes such as shorter main roots, massive lateral root growth, higher leaf and root biomass, and increased anthocyanin levels. In a variety of cocultivation assays, it was shown that these altered phenotypes are caused by fungal CO2, volatile organic compounds, and soluble compounds, notably astins. Astins A, C, and G induced plant growth when they were individually included in the medium. In return, A. thaliana stimulates the fungal astin C production during cocultivation. Taken together, our results indicate a bilateral interaction between the fungus and the plant. A stress response in plants is induced by fungal metabolites while plant stress hormones induced astin C production of the fungus. Interestingly, our results not only show unidirectional influence of the fungus on the plant but also vice versa. The plant is able to influence growth and secondary metabolite production in the endophyte, even when both organisms do not live in close contact, suggesting the involvement of volatile compounds.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Antitumor astins originate from the fungal endophyte Cyanodermella asteris living within the medicinal plant Aster tataricus.

Medicinal plants are a prolific source of natural products with remarkable chemical and biological properties, many of which have considerable remedial benefits. Numerous medicinal plants are suffering from wildcrafting, and thus biotechnological production processes of their natural products are urgently needed. The plant Aster tataricus is widely used in traditional Chinese medicine and contains unique active ingredients named astins. These are macrocyclic peptides showing promising antitumor activities and usually containing the highly unusual moiety 3,4-dichloroproline. The biosynthetic origins of astins are unknown despite being studied for decades. Here we show that astins are produced by the recently discovered fungal endophyte Cyanodermella asteris. We were able to produce astins in reasonable and reproducible amounts using axenic cultures of the endophyte. We identified the biosynthetic gene cluster responsible for astin biosynthesis in the genome of C. asteris and propose a production pathway that is based on a nonribosomal peptide synthetase. Striking differences in the production profiles of endophyte and host plant imply a symbiotic cross-species biosynthesis pathway for astin C derivatives, in which plant enzymes or plant signals are required to trigger the synthesis of plant-exclusive variants such as astin A. Our findings lay the foundation for the sustainable biotechnological production of astins independent from aster plants.

Astin C Production by the Endophytic Fungus Cyanodermella asteris in Planktonic and Immobilized Culture Conditions.

The fungal endophyte Cyanodermella asteris (C. asteris) has been recently isolated from the medicinal plant Aster tataricus (A. tataricus). This fungus produces astin C, a cyclic pentapeptide with anticancer and anti-inflammatory properties. The production of this secondary metabolite is compared in immobilized and planktonic conditions. For immobilized cultures, a stainless steel packing immersed in the culture broth is used as a support. In these conditions, the fungus exclusively grows on the packing, which provides a considerable advantage for astin C recovery and purification. C. asteris metabolism is different according to the culture conditions in terms of substrate consumption rate, cell growth, and astin C production. Immobilized-cell cultures yield a 30% increase of astin C production, associated with a 39% increase in biomass. The inoculum type as spores rather than hyphae, and a pre-inoculation washing procedure with sodium hydroxide, turns out to be beneficial both for astin C production and fungus development onto the support. Finally, the influence of culture parameters such as pH and medium composition on astin C production is evaluated. With optimized culture conditions, astin C yield is further improved reaching a five times higher final specific yield compared to the value reported with astin C extraction from A. tataricus (0.89 mg g-1 and 0.16 mg g-1 respectively).

Linking secondary metabolites to biosynthesis genes in the fungal endophyte Cyanodermella asteris: The anti-cancer bisanthraquinone skyrin.

Fungal aromatic polyketides display a very diverse and widespread group of natural products. Due to their excellent light absorption properties and widely studied biological activities, they offer numerous application for food, textile and pharmaceutical industry. The biosynthetic pathways of fungal aromatic polyketides usually involve a set of successive enzymes, in which a non-reductive polyketide synthase iteratively catalyzes the essential assembly of simple building blocks into (often polycyclic) aromatic compounds. However, only a limited number of such pathways have been described so far and further elucidation of the individual biosynthetic steps is needed to fully exploit the biotechnological and medicinal potential of these compounds. Here, we identified the bisanthraquinone skyrin as the main pigment of the fungus Cyanodermella asteris, an endophyte that has recently been isolated from the traditional Chinese medicinal plant Aster tataricus. The genome of C. asteris was sequenced, assembled and annotated, which enables first insights into a genome from a non-lichenized member of the class Lecanoromycetes. Genetic and in silico analyses led to the identification of a gene cluster of five genes suggested to encode the enzymatic pathway for skyrin. Our study is a starting point for rational pathway engineering in order to drive the production towards higher yields or more active derivatives. Moreover, our investigations revealed a large potential of secondary metabolite production in C. asteris as well as in all Lecanoromycetes of which genomes were available. These findings convincingly emphasize that Lecanoromycetes are prolific producers of secondary metabolites.

Polyketide Bioderivatization Using the Promiscuous Acyltransferase KirCII.

During polyketide biosynthesis, acyltransferases (ATs) are the essential gatekeepers which provide the assembly lines with precursors and thus contribute greatly to structural diversity. Previously, we demonstrated that the discrete AT KirCII from the kirromycin antibiotic pathway accesses nonmalonate extender units. Here, we exploit the promiscuity of KirCII to generate new kirromycins with allyl- and propargyl-side chains in vivo, the latter were utilized as educts for further modification by "click" chemistry.

The cyclochlorotine mycotoxin is produced by the nonribosomal peptide synthetase CctN in Talaromyces islandicus ('Penicillium islandicum').

Talaromyces islandicus ('Penicillium islandicum') is a widespread foodborne mold that produces numerous secondary metabolites, among them potent mycotoxins belonging to different chemical classes. A notable metabolite is the hepatotoxic and carcinogenic pentapeptide cyclochlorotine that contains the unusual amino acids ß-phenylalanine, 2-aminobutyrate and 3,4-dichloroproline. Although the chemical structure has been known for over five decades, nothing is known about the biosynthetic pathway of cyclochlorotine. Bioinformatic analysis of the recently sequenced genome of T. islandicus identified a wealth of gene clusters potentially coding for the synthesis of secondary metabolites. Here, we show by RNA interference-mediated gene silencing that a nonribosomal peptide synthetase, CctN, is responsible for the synthesis of cyclochlorotine. Moreover, we identified novel cyclochlorotine chemical variants, whose production also depended on cctN expression. Surprisingly, the halogenase required for cyclochlorotine biosynthesis is not encoded in the cct cluster. Nonetheless, our findings enabled us to propose a detailed model for cyclochlorotine biosynthesis. In addition, comparative genomics revealed that cct-like clusters are present in all of the sequenced Talaromyces strains indicating a high prevalence of cyclochlorotine production ability.

Draft genome sequence of Talaromyces islandicus ("Penicillium islandicum") WF-38-12, a neglected mold with significant biotechnological potential.

Talaromyces (Penicillium) islandicus is a common mold found in stored rice or cereals. It has a highly versatile metabolism characterized by the secretion of numerous biopolymer degrading enzymes, mycotoxins, and anthraquinones that altogether offer a broad range of potential industrial applications. Here, we report the draft genome sequence of Talaromyces islandicus, which provides the basis of a biotechnological usage of this species.