Discovery and Activation of the Cryptic Cluster from Aspergillus sp. CPCC 400735 for Asperphenalenone Biosynthesis

Abstract

Postgenomic analysis manifested that filamentous fungi contain numerous natural product biosynthetic gene clusters in their genome, yet most clusters remain cryptic or down-regulated. Herein, we report the successful manipulation of strain Aspergillus sp. CPCC 400735 that enables its genetic engineering via targeted overexpression of pathway-specific transcriptional regulator AspE. The down-regulated metabolic pathway encoded by the biosynthetic gene cluster asp was successfully up-activated. Analyses of mutant Ai-OE::aspE extracts led to isolation and characterization of 13 asperphenalenone derivatives, of which 11 of them are new compounds. All of the asperphenalenones exhibited conspicuous anti-influenza A virus effects with IC50 values of 0.45–2.22 μM. Additionally, their identification provided insight into biosynthesis of asperphenalenones and might benefit studies of downstream combinatorial biosynthesis. Our study further demonstrates the effective application of targeted overexpressing pathway-specific activator and novel metabolite discovery in microorganisms. These will accelerate the exploitation of the untapped resources and biosynthetic capability in filamentous fungi.

Genomic and Transcriptomic Approaches Provide a Predictive Framework for Sesquiterpenes Biosynthesis in Desarmillaria tabescens CPCC 401429

“…Genomic DNA from Desarmillaria was isolated using HP Fungal DNA Kit (Omega, Guangzhou, China) according to the manufacturer's instructions. Further purification was conducted using RNase digestion, chloroform extraction, and isopropanol precipitation [29]. The high molecular weight gDNA was washed twice with 75% ethanol, and dissolved in 200 µL RNase-free water.…”

“…The oligonucleotide primers used for constructing the genes are listed in Table 1. PCR reactions were performed using Q5 High-Fidelity DNA Polymerase (New England Biolabs) according to the manufacturer's protocol at an annealing temperature of 56 • C. In vivo yeast recombination cloning strategy was performed using a Frozen-EZ Yeast Transformation II Kit™ (Zymo Research) where competent cell S. cerevisiae BJ5464 was transformed with the individual DNA fragment (PCR product) of the respective STS gene and linearized vector backbone derived from YET plasmid [28,29]. Yeast transformants grown on synthetic dropout agar plate lacking tryptophan were screened by diagnostic PCR.…”

Mining and characterization of the PKS–NRPS hybrid for epicoccamide A: a mannosylated tetramate derivative from Epicoccum sp. CPCC 400996

“…Meanwhile, we could now easily access to the sequenced genome of targeted microorganism whose metabolites have not been, or rarely, characterized. Genome-guided mining approach is usually conducted to set up the linking between secondary metabolites identification and targeted biosynthetic gene clusters (BGCs) verification [ 11 , 12 ]. Also, previous studies have demonstrated that genome mining is an effective approach that facilitated exploitation for new metabolites with fascinating structures, and numerous BGCs were investigated [ 10 , 13 , 14 ].…”