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Airborne fungal spores are a major cause of fungal diseases in humans, animals, and plants as well as contamination of foods. Previous studies found a variety of regulators including VosA, VelB, WetA, and SscA for sporogenesis and the long-term viability in Aspergillus nidulans. To gain a mechanistic understanding of the complex regulatory mechanisms in asexual spores, here, we focused on the relationship between VosA and SscA using comparative transcriptomic analysis and phenotypic studies. The ΔsscA ΔvosA double-mutant conidia have lower spore viability and stress tolerance compared to the ΔsscA or ΔvosA single mutant conidia. Deletion of sscA or vosA affects chitin levels and mRNA levels of chitin biosynthetic genes in conidia. In addition, SscA and VosA are required for the dormant state of conidia and conidial germination by modulating the mRNA levels of the cytoskeleton and development-associated genes. Overall, these results suggest that SscA and VosA play interdependent roles in governing spore maturation, dormancy, and germination in A. nidulans., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Vibrio cholerae is a facultative pathogen that primarily occupies marine environments. In this niche, V. cholerae commonly interacts with the chitinous shells of crustacean zooplankton. As a chitinolytic microbe, V. cholerae degrades insoluble chitin into soluble oligosaccharides. Chitin oligosaccharides serve as both a nutrient source and an environmental cue that induces a strong transcriptional response in V. cholerae. Namely, these oligosaccharides induce the chitin sensor, ChiS, to activate the genes required for chitin utilization and horizontal gene transfer by natural transformation. Thus, interactions with chitin impact the survival of V. cholerae in marine environments. Chitin is a complex carbon source for V. cholerae to degrade and consume, and the presence of more energetically favorable carbon sources can inhibit chitin utilization. This phenomenon, known as carbon catabolite repression (CCR), is mediated by the glucose-specific Enzyme IIA (EIIAGlc) of the phosphoenolpyruvate-dependent phosphotransferase system (PTS). In the presence of glucose, EIIAGlc becomes dephosphorylated, which inhibits ChiS transcriptional activity by an unknown mechanism. Here, we show that dephosphorylated EIIAGlc interacts with ChiS. We also isolate ChiS suppressor mutants that evade EIIAGlc-dependent repression and demonstrate that these alleles no longer interact with EIIAGlc. These findings suggest that EIIAGlc must interact with ChiS to exert its repressive effect. Importantly, the ChiS suppressor mutations we isolated also relieve repression of chitin utilization and natural transformation by EIIAGlc, suggesting that CCR of these behaviors is primarily regulated through ChiS. Together, our results reveal how nutrient conditions impact the fitness of an important human pathogen in its environmental reservoir., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Green et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Chitin, a major component of insect cuticles, plays a critical role in insect molting and morphogenesis. Thus, coordination of chitin remodeling during insect development requires tight transcriptional control of the chitin metabolism genes involved in chitin synthesis, assembly and degradation. However, the molecular mechanism underlying transcriptional coordination of chitin metabolism genes during beetle development is not yet completely understood., Results: We cloned the full-length cDNA encoding hormone receptor 3 (TcHR3) from Tribolium castaneum and showed a critical role of TcHR3 in modulating chitin metabolism gene expression during molting. Genome-wide transcriptome analysis of HR3-deficient old larvae using RNA sequencing analysis revealed a positive correlation between TcHR3 and transcription of chitin metabolism genes involved in chitin synthesis and degradation. In addition, HR3 overexpression significantly induced the gene promoter activity of N-acetylglucosaminidase 1 (NAG1) involved in chitin degradation and UDP-N-acetylglucosamine pyrophosphorylase 1 (UAP1) involved in chitin synthesis. Chromatin immunoprecipitation analysis revealed that HR3 could directly bind to HR3-response element of NAG1 and UAP1 promoters. Finally, HR3-deficient late instar larvae and prepupae exhibited defects in larval-larval and larval-pupal molting, respectively, leading to eventual larval death because developing larvae were trapped inside the old cuticle as a result of abnormal chitin metabolism., Conclusion: TcHR3 is a transcriptional regulator of chitin metabolic genes for molting of T. castaneum. Controlling the molting system by TcHR3 might be a new management strategy for selective control of red flour beetle infestation. © 2022 Society of Chemical Industry., (© 2022 Society of Chemical Industry.)

Chitin is a kind of insoluble structural polysaccharide and plays different roles in different species. In crustaceans, it forms the structural components in the exoskeleton. In our previous studies, novel mud crab hybrids have been produced from the interspecific hybridization of Scylla serrata ♀ × S. paramamosain ♂. Some of the hybrid crabs have been found to be morphologically (eyestalk) abnormal, but the genetic mechanism remains unknown. To address this question, we performed whole-transcriptome RNA sequencing on the control group (normal hybrids), abnormal hybrids, and S. paramamosain to uncover the genetic basis underlying this morphological abnormality. A total of 695 mRNAs, 10 miRNAs, 44 circRNAs, and 1957 lncRNAs were differentially expressed between normal and abnormal hybrids. Several differentially expressed genes (DEGs) associated with chitin and cuticle metabolism were identified, including chitin synthase, chitinase, chitin deacetylase, β-N-acetylglucosaminidase, β-1,4-endoglucanase, N-alpha-acetyltransferase, cuticle proprotein, early cuticle protein, and arthrodial cuticle protein. Functional analysis showed that DE miRNAs, DE circRNAs, DE lncRNAs, and lncRNA/circRNA-miRNA-mRNA network were enriched in pathways related to the amino acid, carbohydrate, and glycogen metabolism. Considering the importance of the chitin and cuticle in exoskeleton formation, it can be concluded that the changes in the chitin and cuticle biosynthesis might have caused the eyestalk abnormality in hybrid crabs. These findings can lay the solid foundation for a better understanding of the important roles of chitin and cuticle related genes and the development of hybridization techniques in crustaceans., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Chitooligosaccharides (COSs) have a widespread range of biological functions and an incredible potential for various pharmaceutical and agricultural applications. Although several physical, chemical, and biological techniques have been reported for COSs production, it is still a challenge to obtain structurally defined COSs with defined polymerization (DP) and acetylation patterns, which hampers the specific characterization and application of COSs. Herein, we achieved the de novo production of structurally defined COSs using combinatorial pathway engineering in Bacillus subtilis. Specifically, the COSs synthase NodC from Azorhizobium caulinodans was overexpressed in B. subtilis, leading to 30 ± 0.86 mg/L of chitin oligosaccharides (CTOSs), the homo-oligomers of N-acetylglucosamine (GlcNAc) with a well-defined DP lower than 6. Then introduction of a GlcNAc synthesis module to promote the supply of the sugar acceptor GlcNAc, reduced CTOSs production, which suggested that the activity of COSs synthase NodC and the supply of sugar donor UDP-GlcNAc may be the limiting steps for CTOSs synthesis. Therefore, 6 exogenous COSs synthase candidates were examined, and the nodCM from Mesorhizobium loti yielded the highest CTOSs titer of 560 ± 16 mg/L. Finally, both the de novo pathway and the salvage pathway of UDP-GlcNAc were engineered to further promote the biosynthesis of CTOSs. The titer of CTOSs in 3-L fed-batch bioreactor reached 4.82 ± 0.11 g/L (85.6% CTOS5, 7.5% CTOS4, 5.3% CTOS3 and 1.6% CTOS2), which was the highest ever reported. This is the first report proving the feasibility of the de novo production of structurally defined CTOSs by synthetic biology, and provides a good starting point for further engineering to achieve the commercial production., (Copyright © 2022 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Background: Using in silico sequence analyses, the present study aims to clone and express the gene-encoding sequence of a GH19 chitinase from Enterobacter sp. in Escherichia coli., Methods and Results: The putative open reading frame of a GH19 chitinase from Enterobacter sp. strain EGY1 was cloned and expressed into pGEM®-T and pET-28a (+) vectors, respectively using a degenerate primer. The isolated nucleotide sequence (1821 bp, GenBank accession no.: MK533791.2) was translated to a chiRAM protein (606 amino acids, UniProt accession no.: A0A4D6J2L9). The in silico protein sequence analysis of chiRAM revealed a class I GH19 chitinase: an N-terminus signal peptide (Met 1 -Ala 23 ), a catalytic domain (Val 83 -Glu 347 and the catalytic triad Glu 149 , Glu 171 , and Ser 218 ), a proline-rich hinge region (Pro 414 -Pro 450 ), a polycystic kidney disease protein motif (Gly 465 -Ser 533 ), a C-terminus chitin-binding domain (Ala 553 - Glu 593 ), and conserved class I motifs (NYNY and AQETGG). A three-dimensional model was constructed by LOMETS MODELLER of PDB template: 2dkvA (class I chitinase of Oryza sativa L. japonica). Recombinant chiRAM was overexpressed as inclusion bodies (IBs) (~ 72 kDa; SDS-PAGE) in 1.0 mM IPTG induced E. coli BL21 (DE3) Rosetta strain at room temperature 18 h after induction. Optimized expression yielded active chiRAM with 1.974 ± 0.0002 U/mL, on shrimp colloidal chitin (SCC), in induced E. coli BL21 (DE3) Rosetta cells growing in SB medium. LC-MS/MS identified a band of 72 kDa in the soluble fraction with a 52.3% coverage sequence exclusive to the GH19 chitinase of Enterobacter cloacae (WP_063869339.1)., Conclusions: Although chiRAM of Enterobacter sp. was successfully cloned and expressed in E. coli with appreciable chitinase activity, future studies should focus on minimizing IBs to facilitate chiRAM purification and characterization., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

The discovery of oxidative cleavage of recalcitrant polysaccharides by lytic polysaccharide monooxygenases (LPMOs) has affected the study and industrial application of enzymatic biomass processing. Despite being widespread in fungi, LPMOs belonging to the auxiliary activity (AA) family AA11 have been understudied. While these LPMOs are considered chitin active, some family members have little or no activity toward chitin, and the only available crystal structure of an AA11 LPMO lacks features found in bacterial chitin-active AA10 LPMOs. Here, we report structural and functional characteristics of a single-domain AA11 LPMO from Aspergillus fumigatus, AfAA11A. The crystal structure shows a substrate-binding surface with features resembling those of known chitin-active LPMOs. Indeed, despite the absence of a carbohydrate-binding module, AfAA11A has considerable affinity for α-chitin and, more so, β-chitin. AfAA11A is active toward both these chitin allomorphs and enhances chitin degradation by an endoacting chitinase, in particular for α-chitin. The catalytic activity of AfAA11A on chitin increases when supplying reactions with hydrogen peroxide, showing that, like LPMOs from other families, AfAA11A has peroxygenase activity. These results show that, in stark contrast to the previously characterized AfAA11B from the same organism, AfAA11A likely plays a role in fungal chitin turnover. Thus, members of the hitherto rather enigmatic family of AA11 LPMOs show considerable structural and functional differences and may have multiple roles in fungal physiology., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)