The transcriptional factor Clr-5 is involved in cellulose degradation through regulation of amino acid metabolism in Neurospora crassa.

BACKGROUND: Filamentous fungi are efficient degraders of plant biomass and the primary producers of commercial cellulolytic enzymes. While the transcriptional regulation mechanisms of cellulases have been continuously explored in lignocellulolytic fungi, the induction of cellulase production remains a complex multifactorial system, with several aspects still largely elusive. RESULTS: In this study, we identified a Zn2Cys6 transcription factor, designated as Clr-5, which regulates the expression of cellulase genes by influencing amino acid metabolism in Neurospora crassa during growth on cellulose. The deletion of clr-5 caused a significant decrease in secreted protein and cellulolytic enzyme activity of N. crassa, which was partially alleviated by supplementing with yeast extract. Transcriptomic profiling revealed downregulation of not only the genes encoding main cellulases but also those related to nitrogen metabolism after disruption of Clr-5 under Avicel condition. Clr-5 played a crucial role in the utilization of multiple amino acids, especially leucine and histidine. When using leucine or histidine as the sole nitrogen source, the Δclr-5 mutant showed significant growth defects on both glucose and Avicel media. Comparative transcriptomic analysis revealed that the transcript levels of most genes encoding carbohydrate-active enzymes and those involved in the catabolism and uptake of histidine, branched-chain amino acids, and aromatic amino acids, were remarkably reduced in strain Δclr-5, compared with the wild-type N. crassa when grown in Avicel medium with leucine or histidine as the sole nitrogen source. These findings underscore the important role of amino acid metabolism in the regulation of cellulase production in N. crassa. Furthermore, the function of Clr-5 in regulating cellulose degradation is conserved among ascomycete fungi. CONCLUSIONS: These findings regarding the novel transcription factor Clr-5 enhance our comprehension of the regulatory connections between amino acid metabolism and cellulase production, offering fresh prospects for the development of fungal cell factories dedicated to cellulolytic enzyme production in bio-refineries.

The arabinose transporter MtLat-1 is involved in hemicellulase repression as a pentose transceptor in Myceliophthora thermophila.

BACKGROUND: Filamentous fungi possess an array of secreted enzymes to depolymerize the structural polysaccharide components of plant biomass. Sugar transporters play an essential role in nutrient uptake and sensing of extracellular signal molecules to inhibit or trigger the induction of lignocellulolytic enzymes. However, the identities and functions of transceptors associated with the induction of hemicellulase genes remain elusive. RESULTS: In this study, we reveal that the L-arabinose transporter MtLat-1 is associated with repression of hemicellulase gene expression in the filamentous fungus Myceliophthora thermophila. The absence of Mtlat-1 caused a decrease in L-arabinose uptake and consumption rates. However, mycelium growth, protein production, and hemicellulolytic activities were markedly increased in a ΔMtlat-1 mutant compared with the wild-type (WT) when grown on arabinan. Comparative transcriptomic analysis showed a different expression profile in the ΔMtlat-1 strain from that in the WT in response to arabinan, and demonstrated that MtLat-1 was involved in the repression of the main hemicellulase-encoding genes. A point mutation that abolished the L-arabinose transport activity of MtLat-1 did not impact the repression of hemicellulase gene expression when the mutant protein was expressed in the ΔMtlat-1 strain. Thus, the involvement of MtLat-1 in the expression of hemicellulase genes is independent of its transport activity. The data suggested that MtLat-1 is a transceptor that senses and transduces the molecular signal, resulting in downstream repression of hemicellulolytic gene expression. MtAra-1 protein directly regulated the expression of Mtlat-1 by binding to its promoter region. Transcriptomic profiling indicated that the transcription factor MtAra-1 also plays an important role in expression of arabinanolytic enzyme genes and L-arabinose catabolism. CONCLUSIONS: M. thermophila MtLat-1 functions as a transceptor that is involved in L-arabinose transport and signal transduction associated with suppression of the expression of hemicellulolytic enzyme-encoding genes. The data presented in this study add to the models of the regulation of hemicellulases in filamentous fungi.

High-Field Asymmetric Waveform Ion Mobility Spectrometry Interface Enhances Parallel Reaction Monitoring on an Orbitrap Mass Spectrometer.

High-field asymmetric waveform ion mobility spectrometry (FAIMS) enables gas-phase separations on a chromatographic time scale and has become a useful tool for proteomic applications. Despite its emerging utility, however, the molecular determinants underlying peptide separation by FAIMS have not been systematically investigated. Here, we characterize peptide transmission in a FAIMS device across a broad range of compensation voltages (CVs) and used machine learning to identify charge state and three-dimensional (3D) electrostatic peptide potential as major contributors to peptide intensity at a given CV. We also demonstrate that the machine learning model can be used to predict optimized CV values for peptides, which significantly improves parallel reaction monitoring workflows. Together, these data provide insight into peptide separation by FAIMS and highlight its utility in targeted proteomic applications.