First-class - biosynthesis of 6-MSA and bostrycoidin type I polyketides in Yarrowia lipolytica.

Fungal polyketides are a large group of secondary metabolites, valuable due to their diverse spectrum of pharmacological activities. Polyketide biosynthesis in filamentous fungi presents some challenges: small yield and low-purity titers. To tackle these issues, we switched to the yeast Yarrowia lipolytica, an easily cultivable heterologous host. As an oleaginous yeast, Y. lipolytica displays a high flux of acetyl- and malonyl-CoA precursors used in lipid synthesis. Likewise, acetyl- and malonyl-CoA are the building blocks of many natural polyketides, and we explored the possibility of redirecting this flux toward polyketide production. Despite its promising prospect, Y. lipolytica has so far only been used for heterologous expression of simple type III polyketide synthases (PKSs) from plants. Therefore, we decided to evaluate the potential of Y. lipolytica by targeting the more complex fungal polyketides synthesized by type I PKSs. We employed a CRISPR-Cas9-mediated genome editing method to achieve markerless gene integration of the genes responsible for bostrycoidin biosynthesis in Fusarium solani (fsr1, fsr2, and fsr3) and 6-methylsalicylic acid (6-MSA) biosynthesis in Aspergillus hancockii (6MSAS). Moreover, we attempted titer optimization through metabolic engineering by overexpressing two enzymes, TGL4 and AOX2, involved in lipid ß-oxidation, but we did not observe an effect on polyketide production. With maximum titers of 403 mg/L 6-MSA and 35 mg/L bostrycoidin, the latter being substantially higher than our previous results in Saccharomyces cerevisiae (2.2 mg/L), this work demonstrates the potential of Y. lipolytica as a platform for heterologous production of complex fungal polyketides.

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.

A new vector system for targeted integration and overexpression of genes in the crop pathogen Fusarium solani.

BACKGROUND: Besides their ability to produce several interesting bioactive secondary metabolites, members of the Fusarium solani species complex comprise important pathogens of plants and humans. One of the major obstacles in understanding the biology of this species complex is the lack of efficient molecular tools for genetic manipulation. RESULTS: To remove this obstacle we here report the development of a reliable system where the vectors are generated through yeast recombinational cloning and inserted into a specific site in F. solani through Agrobacterium tumefaciens-mediated transformation. As proof-of-concept, the enhanced yellow fluorescent protein (eYFP) was inserted in a non-coding genomic position of F. solani and subsequent analyses showed that the resulting transformants were fluorescent on all tested media. In addition, we cloned and overexpressed the Zn(II)2Cys6 transcriptional factor fsr6 controlling mycelial pigmentation. A transformant displayed deep red/purple pigmentation stemming from bostrycoidin and javanicin. CONCLUSION: By creating streamlined plasmid construction and fungal transformation systems, we are now able to express genes in the crop pathogen F. solani in a reliable and fast manner. As a case study, we targeted and activated the fusarubin (PKS3: fsr) gene cluster, which is the first case study of secondary metabolites being directly associated with the responsible gene cluster in F. solani via targeted activation. The system provides an approach that in the future can be used by the community to understand the biochemistry and genetics of the Fusarium solani species complex, and is obtainable from Addgene catalog #133094.

Fusarubin and Anhydrofusarubin Isolated from A Cladosporium Species Inhibit Cell Growth in Human Cancer Cell Lines.

Cladosporium species are endophytic fungi that grow on organic matter and are considered food contaminants. The anti-microbial and anti-tumor naphthoquinones fusarubin (FUS) and anhydrofusarubin (AFU) were isolated using column chromatography from a Cladosporium species residing inside Rauwolfia leaves. The impact of FUS and AFU on cell growth was assessed in acute myeloid leukemia (OCI-AML3) and other hematologic tumor cell lines (HL-60, U937, and Jurkat). Treatment with FUS or AFU reduced the number of OCI-AML3 cells as evaluated by a hemocytometer. Flow cytometry analyses showed that this effect was accompanied by diverse impairments in cell cycle progression. Specifically, FUS (20 or 10 µg/mL significantly decreased the percentage of cells in S phase and increased the percentage of cells in G2/M phase, whereas AFU increased the percentage of cells in G0/G1 phase (50 and 25 µg/mL) and decreased the percentage of cells in S (50 µg/mL) and G2/M (50 and 25 µg/mL) phases. Both substances significantly increased apoptosis at higher concentrations. The effects of FUS were more potent than those of AFU, with FUS up-regulating p21 expression in a p53-dependent manner, as detected by Western blot analyses, likely the consequence of decreased ERK phosphorylation and increased p38 expression (both of which increase p21 stability). FUS also decreased Akt phosphorylation and resulted in increased Fas ligand production and caspase-8/3-dependent apoptosis. These results suggest that FUS and AFU inhibit proliferation and increase apoptosis in cell lines derived from hematological cancers.

Cytotoxic and antibacterial naphthoquinones from an endophytic fungus, Cladosporium sp.

OBJECTIVE: Endophytes have the potential to synthesize various bioactive secondary metabolites. The aim of the study was to find new cytotoxic and antibacterial metabolites from endophytic fungus, Cladosporium sp. isolated from the leaves of Rauwolfia serpentina (L.) Benth. ex Kurz. (Fam: Apocyanaceae). MATERIALS AND METHODS: The endophytic fungus was grown on potato dextrose agar medium and extracted using ethyl acetate. Secondary metabolites were isolated by chromatographic separation and re-crystallization, and structures were confirmed by 1H NMR, 13C NMR and mass spectroscopic data. The cytotoxicity was determined by WST-1 assay and brine shrimp lethality bioassay, while antibacterial activity was assessed by disc diffusion method. RESULTS: Two naphthoquinones, namely anhydrofusarubin (1) and methyl ether of fusarubin (2), were isolated from Cladosporium sp. The isolated compounds 1 and 2, by WST-1 assay against human leukemia cells (K-562) showed potential cytotoxicity with IC50 values of 3.97 µg/mL and 3.58 µg/mL, respectively. Initial screening of crude ethyl acetate extract and column fractions F-8 and F-10 exhibited noticeable cytotoxicity to brine shimp nauplii with LC50 values of 42.8, 1.2 and 2.1 µg/mL, respectively. Moreover, the isolated compound 2 (40 µg/disc) showed prominent activities against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Bacillus megaterium with an average zone of inhibition of 27 mm, 25 mm, 24 mm and 22 mm, respectively and the activities were compared with kanamycin (30 µg/disc). CONCLUSION: Our findings indicate that anhydrofusarubin (1) and methyl ether of fusarubin (2) might be useful lead compounds to develop potential cytotoxic and antimicrobial drugs.