Manipulation of the Global Regulator mcrA Upregulates Secondary Metabolite Production in Aspergillus wentii Using CRISPR-Cas9 with In Vitro Assembled Ribonucleoproteins

Abstract

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.

Establishment, optimization, and application of genetic technology in Aspergillus spp.

“…Even mycotoxins synthesis, which is considered harmful and regulated comprehensively through complex pathways such as anabolism and catabolism, can be controlled in the cascade of external environmental factors ( Gao et al, 2021 ), the knockdown of key genes in mycotoxin biosynthesis gene clusters or cross-regulatory pathways can prevent and control harmful mycotoxin synthesis from the source. Overexpression of specific genes can activate silencing pathways and may discover more useful secondary metabolites ( Yuan et al, 2022 ). Therefore, the study of Aspergillus inevitably needs to go deep into the genetic level, the exploration of mechanisms and modification of gene functions can be realized through gene editing technologies by pathways reconstruction or activating silent pathways.…”

Advances and Challenges in CRISPR/Cas-Based Fungal Genome Engineering for Secondary Metabolite Production: A Review

“…The application of this RNP-based system for A. fumigatus gene editing provided a simple and universal way to tackle the problem of virulence and antifungal drug resistance in multiple clinical isolates of this strain. An in vitro CRISPR/Cas9 system was established in wild-type Aspergillus wentii to delete a negative transcriptional regulator, mcrA , which is a master regulator of SM clusters, resulting in the enhanced production of a range of new SMs due to the activation of a polyketide synthase (PKS), BGC [ 19 , 60 ]. RNP complexes of modified Cas9 nuclease and pairs of single guide RNAs were used in Epichloë species to eliminate the entire ergot alkaloid biosynthesis cluster, which avoided the production of SMs that are toxic to livestock [ 61 ].…”

Sigma Factor Engineering in Actinoplanes sp. SE50/110: Expression of the Alternative Sigma Factor Gene ACSP50_0507 (σHAs) Enhances Acarbose Yield and Alters Cell Morphology

“…For other species, it was shown that the manipulation of global transcriptional regulators can be used to achieve increased metabolite production. The manipulation can be performed using different approaches, e.g., with gene deletion, using evolved transcriptional regulators or overexpression [ 15 , 16 , 17 ]. Sigma factors were shown to be superior targets to manipulate metabolite production, as they control larger transcriptional networks.…”