The F-box protein gene exo - 1 is a target for reverse engineering enzyme hypersecretion in filamentous fungi

Abstract

Carbohydrate active enzymes (CAZymes) are vital for the lignocellulose-based biorefinery. The development of hypersecreting fungal protein production hosts is therefore a major aim for both academia and industry. However, despite advances in our understanding of their regulation, the number of promising candidate genes for targeted strain engineering remains limited. Here, we resequenced the genome of the classical hypersecreting Neurospora crassa mutant exo-1 and identified the causative point of mutation to reside in the F-box protein–encoding gene, NCU09899. The corresponding deletion strain displayed amylase and invertase activities exceeding those of the carbon catabolite derepressed strain Δcre-1, while glucose repression was still mostly functional in Δexo-1. Surprisingly, RNA sequencing revealed that while plant cell wall degradation genes are broadly misexpressed in Δexo-1, only a small fraction of CAZyme genes and sugar transporters are up-regulated, indicating that EXO-1 affects specific regulatory factors. Aiming to elucidate the underlying mechanism of enzyme hypersecretion, we found the high secretion of amylases and invertase in Δexo-1 to be completely dependent on the transcriptional regulator COL-26. Furthermore, misregulation of COL-26, CRE-1, and cellular carbon and nitrogen metabolism was confirmed by proteomics. Finally, we successfully transferred the hypersecretion trait of the exo-1 disruption by reverse engineering into the industrially deployed fungus Myceliophthora thermophila using CRISPR-Cas9. Our identification of an important F-box protein demonstrates the strength of classical mutants combined with next-generation sequencing to uncover unanticipated candidates for engineering. These data contribute to a more complete understanding of CAZyme regulation and will facilitate targeted engineering of hypersecretion in further organisms of interest.

Chromatin-immunoprecipitation reveals the PnPf2 transcriptional network controlling effector-mediated virulence in a fungal pathogen of wheat

“…The PnCreA orthologue of N. crassa Cre-1 was also investigated, following the striking observation that the N. crassa Cre-1 CCR element (5′-TSYGGGG-3') was enriched in PnPf2-regulated gene promoters (13). Furthermore, Cre-1 and the PnPf2 orthologue Col-26 are both key components of a transcriptional network controlling CAZyme production in N. crassa (22)(23)(24)43). Here, the creA_KO strain displayed an enhanced capacity to utilise starch, which was moderately impaired in the pf2ko mutant (Fig.…”

“…Analogous carbohydrate regulatory roles have been attributed in the saprophytic fungi Neurospora crassa and Trichoderma reesei (20,21). In N. crassa, the putative orthologue Col-26 is a critical component within a signalling-network that responds to glucose availability and promotes the expression of CAZymes for plant cell-wall degradation (22)(23)(24). There is a strong association between CAZyme gene content and plant-pathogenic lifestyles (25).…”

Combined transcriptome and proteome analysis of Bcfrp1 involved in regulating the biosynthesis of abscisic acid and growth in Botrytis cinerea TB-31

“…lycopersici by random insertion, and identified that the insertion site occurred in a gene encoding an F-box protein and named Frp1 ( Duyvesteijn et al, 2005 ). It was reported that F-box protein (FBP) is part of SCFs (Skp1-cullin-F-box protein ligase) complexes and linked to the Skp1 protein through the F-box ( Gabriel et al, 2021 ). Of course, F-box proteins function as non-SCF complexes, too ( Sadat et al, 2021 ; Wu et al, 2022 ).…”

Revisiting 2-Deoxyglucose Resistance in Neurospora crassa: Insights into Transcriptional Regulation of Hexose Transporters, Sugar Metabolism and Possible Modulation by Mating Type.

“…Of these, dgr-3 was shown to be allelic to rco-3 (primarily a conidiation defective mutant, bearing similarity to a putative sugar transporter similar to snf-2 and rgt-3). More recently, the dgr (L1) allelic with dgr-2 was shown to be contain a mutation in an F-box containing transcription factor exo-1 (Gabriel et al 2021). Furthermore, a combination of exo-1 (S11L) as well as ∆col-26, has been shown to be resistant to the rare sugar L-Sorbose (Hirai et al 2022) The inhibition of glycolysis has long been an important strategy associated with cancer treatment (Luengo, Gui, and Vander Heiden 2017), leading to the widespread use 2-DG, though primarily as a supporting molecule.…”