Azo-CM-Cellulose (Powder)

New chromogenic substrates have been developed for the quantitative assay of alpha-amylase and (1→4)-β-D-glucanase. These were prepared by chemically modifying amylose or cellulose before dyeing, to increase solubility. After dyeing, the substrates were either soluble or could be readily dispersed to form fine, gelatinous suspensions. Assays based on the use of these substrates are sensitive and highly specific for either alpha-amylase or (1→4)-β-D-glucanase. The method of preparation can also be applied to obtain substrates for other endo-hydrolases.

Filamentous fungi are important producers of enzymes and secondary metabolites. The industrial thermophilic species, Thermothelomyces thermophilus, is closely related to the model fungus, Neurospora crassa. A critical aspect of the filamentous fungal life cycle is the production of asexual spores (conidia), which are regulated by various stimuli, including nutrient availability. Several species of fungi, including T. thermophilus, produce conidia under submerged fermentation conditions, which can be detrimental to product yields. In this study, transcriptional profiling of T. thermophilus was used to map changes during asexual development in submerged cultures, which revealed commonalities of regulation between T. thermophilus and N. crassa. We further identified a transcription factor, res1, whose deletion resulted in a complete loss of conidia production under fermentation conditions, but which did not affect conidiation on plates. Under fermentation conditions, the ∆res1 deletion strain showed increased biomass production relative to the wild-type strain, indicating that the manipulation of res1 in T. thermophilus has the potential to increase productivity in industrial settings. Overexpression of res1 caused a severe growth defect and early conidia production on both plates and in submerged cultures, indicating res1 overexpression can bypass regulatory aspects associated with conidiation on plates. Using chromatin-immunoprecipitation sequencing, we identified 35 target genes of Res1, including known conidiation regulators identified in N. crassa, revealing common and divergent aspects of asexual reproduction in these two species.

Classical fungal mutant strains obtained by mutagenesis have helped to elucidate fundamental metabolic pathways in the past. In the filamentous fungus Neurospora crassa, the gluc-1 strain was isolated long ago and characterized by its low level of β-glucosidase activity, which is essential for the degradation of cellulose, the most abundant biopolymer on Earth and the main polymeric component of the plant cell wall. Based on genomic resequencing, we hypothesized that the causative mutation resides in the β-glucosidase gene gh3-3 (bgl6, NCU08755). In this work, growth patterns, enzymatic activities and sugar utilization rates were analyzed in several mutant and overexpression strains related to gluc-1 and gh3-3. In addition, different mutants affected in the degradation and transport of cellobiose were analyzed. While overexpression of gh3-3 led to the recovery of β-glucosidase activity in the gluc-1 mutant, as well as normal utilization of cellobiose, the full gene deletion strain Δgh3-3 was found to behave differently than gluc-1 with lower secreted β-glucosidase activity, indicating a dominant role of the amino acid substitution in the point mutated gh3-3 gene of gluc-1. Our results furthermore confirm that GH3-3 is the major extracellular β-glucosidase in N. crassa and demonstrate that the two cellodextrin transporters CDT-1 and CDT-2 are essential for growth on cellobiose when the three main N. crassa β-glucosidases are absent. Overall, these findings provide valuable insight into the mechanisms of cellulose utilization in filamentous fungi, being an essential step in the efficient production of biorefinable sugars from agricultural and forestry plant biomass.

Cellulose is a renewable feedstock for green industry. It is therefore important to develop a technique to construct a host with a high cellulolytic efficiency to digest cellulose. In this study, we developed a convenient host-engineering technique to adjust the expression levels of heterologous genes in the host by promoter rearrangement and gene copy number adjustment. Using genes from different glycoside hydrolase (GH) families including GH2, GH3, GH5, GH6, GH7, and GH12 from Aspergillus niger, Trichoderma reesei, and Neocallimastix patriciarum, we constructed a cellulolytic Kluyveromyces marxianus with eight cellulase gene-cassettes that produced a cellulase cocktail with a high cellulolytic efficiency, leading to a significant reduction in enzyme cost in a rice straw saccharification process. Our technique can be used to design a host that can efficiently convert biomass feedstock to biofuel.

Background: Several protists have evolved the ability to perforate the cell walls of algae and fungi to specifically feed on their cell contents. These phagotrophic “protoplast feeders” represent an interesting mechanistic intermediate between predators and parasites and pose a number of cell biological questions. Although their fascinating feeding behaviour has been observed for the last 150 years, it is still unknown how protoplast feeders produce the well-defined and species-specific perforations in biochemically diverse cell walls. Differential expression analyses of the algivorous flagellate Orciraptor agilis (Viridiraptoridae, Cercozoa, Rhizaria) suggested the involvement of a highly expressed putative glycoside hydrolase of family GH5_5. To assess the importance of this carbohydrate-active enzyme in the feeding act of Orciraptor, we recombinantly produced its catalytic domain and studied the enzymatic activity, cellular localisation and function. Results: The GH5_5 catalytic domain from Orciraptor showed pronounced activity on soluble cellulose derivatives and mixed-linkage glucans, with reaction optima comparable to known GH5_5 representatives. Crystalline cellulose was not digested by the enzyme, which suggests a typical endocellulase activity. Immunocytochemistry with a polyclonal antibody raised against the GH5_5 domain revealed that the native endocellulase localises to the contact zone of Orciraptor and the algal cell wall (= perforation zone) and to intracellular granules, which were enriched during attack. Furthermore, the anti-GH5_5 antibody applied to live cells significantly reduced the feeding success of Orciraptor. The cells attacked the algae, which, however, resulted in numerous incomplete perforations. Conclusions: Our experimental data from enzymatic assays, immunocytochemistry and inhibition experiments strongly suggest a key role of the GH5_5 endocellulase in cell wall dissolution by Orciraptor agilis. With that, we provide evidence that the well-defined perforations produced by protoplast feeders are caused by extracellular carbohydrate-active enzymes and made a first step towards establishing the molecular basis of a fascinating, yet poorly understood microbial feeding strategy.

The thermophilic fungus Myceliophthora thermophila has been used to produce industrial enzymes and biobased chemicals. In saprotrophic fungi, the mechanisms regulating cellulase production have been studied, which revealed the involvement of multiple transcription factors. However, in M. thermophila, the transcription factors influencing cellulase gene expression and secretion remain largely unknown. In this study, we identified and characterized a novel cellulase regulator (MtTRC-1) in M. thermophila through a combination of functional genomics and genetic analyses. Deletion of Mttrc-1 resulted in significantly decreased cellulase production and activities. Transcriptome analysis revealed downregulation of not only the encoding genes of main cellulases but also the transcriptional regulator MtHAC-1 of UPR pathway after disruption of MtTRC-1 under cellulolytic induction conditions. Herein, we also characterized the ortholog of the yeast HAC1p in M. thermophila. We show that Mthac-1 mRNA undergoes an endoplasmic reticulum (ER) stress-induced splicing by removing a 23-nucleotide (nt) intron. Notably, the protein secretion on cellulose was dramatically impaired by the deletion of MtHAC-1. Moreover, the colonial growth on various carbon sources was defective in the absence of MtHAC-1. Electrophoretic mobility shift assays and chromatin immunoprecipitation assays verified MtTRC-1 regulates the transcription of Mthac-1 and the major cellulase gene Mtcbh-1 by binding directly to the promoters in vitro and in vivo. Furthermore, DNase I footprinting assays identified the putative consensus binding site (5′-GNG/C-3′). These results revealed the importance of MtTRC-1 for positively regulating cellulase production. This finding has clarified the complex regulatory pathways involved in cellulolytic enzyme production.

Myceliophthora thermophila is a thermophilic fungus with great potential in biorefineries and biotechnology. The base editor is an upgraded version of the clustered regularly interspaced short palindromic repeats (CRISPR)-dependent genome-editing tool that introduces precise point mutations without causing DNA double-strand breaks (DSBs) and has been used in various organisms but rarely in filamentous fungi, especially thermophilic filamentous fungi. Here, for the first time, we constructed three cytosine base editors (CBEs) in M. thermophila, namely, evolved apolipoprotein B mRNA-editing enzyme catalytic subunit 1 (APOBEC1) cytosine base editor 4 max (Mtevo-BE4max), bacteriophage Mu Gam protein cytosine base editor 4 max (MtGAM-BE4max), and evolved CDA1 deaminase cytosine base editor (Mtevo-CDA1), and efficiently inactivated genes by precisely converting three codons (CAA, CAG, and CGA) into stop codons without DSB formation. The Mtevo-CDA1 editor with up to 92.6% editing efficiency is a more suitable tool for cytosine base editing in thermophilic fungi. To investigate the function of each motif of the cellulase transcription factor M. thermophila CLR-2 (MtCLR-2), we used the Mtevo-CDA1 editor. The fungal-specific motif of MtCLR-2 was found to be strongly involved in cellulase secretion, conidium formation, hyphal branching, and colony formation. Mutation of the fungus-specific motif caused significant defects in these characteristics. Thus, we developed an efficient thermophilic fungus-compatible base-editing system that could also be used for genetic engineering in other relevant filamentous fungi.

Aspergillus niger is a filamentous fungus well known for its ability to produce a wide variety of pectinolytic enzymes, which have many applications in the industry. The transcriptional activator GaaR is induced by 2-keto-3-deoxy-L-galactonate, a compound derived from D-galacturonic acid, and plays a major role in the regulation of pectinolytic genes. The requirement for inducer molecules can be a limiting factor for the production of enzymes. Therefore, the generation of chimeric transcription factors able to activate the expression of pectinolytic genes by using underutilized agricultural residues would be highly valuable for industrial applications. In this study, we used the CRISPR/Cas9 system to generate three chimeric GaaR-XlnR transcription factors expressed by the xlnR promoter by swapping the N-terminal region of the xylanolytic regulator XlnR to that of the GaaR in A. niger. As a test case, we constructed a PpgaX-hph reporter strain to evaluate the alteration of transcription factor specificity in the chimeric mutants. Our results showed that the chimeric GaaR-XlnR transcription factor was induced in the presence of D-xylose. Additionally, we generated a constitutively active GaaR-XlnR V756F version of the most efficient chimeric transcription factor to better assess its activity. Proteomics analysis confirmed the production of several pectinolytic enzymes by ΔgaaR mutants carrying the chimeric transcription factor. This correlates with the improved release of D-galacturonic acid from pectin by the GaaR-XlnR V756F mutant, as well as by the increased L-arabinose release from the pectin side chains by both chimeric mutants under inducing condition, which is required for efficient degradation of pectin.

Plant expression of microbial Cell Wall Degrading Enzymes (CWDEs) is a valuable strategy to produce industrial enzymes at affordable cost. Unfortunately, the constitutive expression of CWDEs may affect plant fitness to variable extents, including developmental alterations, sterility and even lethality. In order to explore novel strategies for expressing CWDEs in crops, the cellobiohydrolase CBM3GH5, from the hyperthermophilic bacterium Caldicellulosiruptor saccharolyticus, was constitutively expressed in N. tabacum by targeting the enzyme both to the apoplast and to the protein storage vacuole. The apoplast targeting failed to isolate plants expressing the recombinant enzyme despite a large number of transformants being screened. On the opposite side, the targeting of the cellobiohydrolase to the protein storage vacuole led to several transgenic lines expressing CBM3GH5, with an enzyme yield of up to 0.08 mg g DW⁻¹ (1.67 Units g DW⁻¹) in the mature leaf tissue. The analysis of CBM3GH5 activity revealed that the enzyme accumulated in different plant organs in a developmental-dependent manner, with the highest abundance in mature leaves and roots, followed by seeds, stems and leaf ribs. Notably, both leaves and stems from transgenic plants were characterized by an improved temperature-dependent saccharification profile.

The efficient use of the yeast Yarrowia lipolytica as a cell factory is hampered by the lack of powerful genetic engineering tools dedicated for the assembly of large DNA fragments and the robust expression of multiple genes. Here we describe the design and construction of artificial chromosomes (ylAC) that allow easy and efficient assembly of genes and chromosomal elements. We show that metabolic pathways can be rapidly constructed by various assembly of multiple genes in vivo into a complete, independent and linear supplementary chromosome with a yield over 90%. Additionally, our results reveal that ylAC can be genetically maintained over multiple generations either under selective conditions or, without selective pressure, using an essential gene as the selection marker. Overall, the ylACs reported herein are game-changing technology for Y. lipolytica, opening myriad possibilities, including enzyme screening, genome studies and the use of this yeast as a previous unutilized bio-manufacturing platform.