Genome sequencing of filamentous
fungi has demonstrated
that most
secondary metabolite biosynthetic gene clusters (BGCs) are silent
under standard laboratory conditions. In this work, we have established
an in vitro CRISPR-Cas9 system in Aspergillus wentii. To activate otherwise silent BGCs, we deleted the negative transcriptional
regulator mcrA. Deletion of mcrA (mcrAΔ) resulted in differential production
of 17 SMs in total when the strain was cultivated on potato dextrose
media (PDA). Nine out of fifteen of these SMs were fully characterized,
including emodin (1), physcion (2), sulochrin
(3), physcion bianthrone (4), 14-O-demethylsulochrin (5), (trans/cis)-emodin bianthrone (6 and 7), and (trans/cis)-emodin
physcion bianthrone (8 and 9). These compounds
were all found to be produced by the same polyketide synthase (PKS)
BGC. We then performed a secondary knockout targeting this PKS cluster
in the mcrAΔ background. The metabolite profile
of the dual-knockout strain revealed new metabolites that were not
previously detected in the mcrAΔ parent strain.
Two additional SMs were purified from the dual-knockout strain and
were characterized as aspergillus acid B (16) and a structurally
related but previously unidentified compound (17). For
the first time, this work presents a facile genetic system capable
of targeted gene editing in A. wentii. This work also illustrates the utility of performing a dual knockout
to eliminate major metabolic products, enabling additional SM discovery.
Filamentous fungi are an essential source of bioactive
mycotoxins.
Recent efforts have focused on developing antifungal agents that are
effective against invasive yeasts, such as Candida spp. By screening fungal strains isolated from regions surrounding
the Chernobyl nuclear power plant disaster for antifungal activity
against Candida albicans, we found
that Aspergillus melleus IMV 01140
produced compounds that inhibited the growth of the yeast. The active
compound produced by A. melleus was
isolated and found to be neoaspergillic acid, a compound that is closely
related to aspergillic acid. While aspergillic acid and its derivatives
have been characterized and were found to have antibacterial and antifungal
properties, neoaspergillic acid has been much less studied. Even though
neoaspergillic acid and related compounds were found to have antibacterial
and antitumoral effects, further investigation into this group of
compounds is limited by challenges associated with large-scale production,
isolation, and purification. The production of neoaspergillic acid
has been shown to require co-cultivation methods or special growth
conditions. In this work, neoaspergillic acid and related compounds
were found to be produced by A. melleus under laboratory growth conditions. The biosynthetic gene cluster
of neoaspergillic acid was predicted using the aspergillic acid gene
cluster as a model. The biosynthetic pathway for neoaspergillic acid
was then confirmed by establishing an in vitro CRISPR-ribonucleoprotein
system to individually delete genes within the cluster. A negative
transcriptional factor, mcrA, was also eliminated
to further improve the production of neoaspergillic acid and the related
compounds for future studies.
a b s t r a c tFocused crawling is increasingly seen as a solution to address the scalability limitations of existing general-purpose search engines, by traversing the Web to only gather pages that are relevant to a specific topic. How to predict the relevance of the unvisited pages pointed to by candidate URLs in the crawling frontier to a given topic is a key issue in the design of focused crawlers. In this paper, we propose a novel approach based on multiple relevance prediction strategies to address this problem. For cross-language crawling, we first introduce a hierarchical taxonomy to describe topics in both English and Chinese. We then present a formal description of the relevance predicting process and discuss four strategies that make use of page contents, anchor texts, URL addresses and link types of Web pages, respectively, to evaluate the relevance more accurately, in which we propose a particular strategy using Chinese URL addresses to estimate the relevance of cross-language Web pages. Finally, we get a new focused crawling algorithm (FCMRPS, Focused Crawling based on Multiple Relevance Prediction Strategies) based on the combination of these strategies and Shark-Search, which is a classic focused crawling algorithm. Experiments show that the FCMRPS is more effective than the traditional algorithms, namely BreadthFirst, Best-First and Shark-Search, in terms of precision and sum of information.
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