Successive mycelial subculturing decreased lignocellulase activity and increased ROS accumulation in Volvariella volvacea.

Strain degradation is a common problem in many artificially-cultivated edible mushrooms. As a fungus with poor tolerance to low-temperature, Volvariella volvacea cannot delay its degradation by long-term low temperature storage like other fungi, so its degradation is particularly severe, which hinders industrial applications. Periodic mycelial subculture is a common storage method for V. volvacea, but excessive subculturing can also lead to strain degeneration. After 20 months of continuous subculturing every 3 days, V. volvacea strains S1-S20 were obtained, and their characteristics throughout the subculture process were analyzed. With increasing number of subculture, the growth rate, mycelial biomass, the number of fruiting bodies and biological efficiency gradually decreased while the production cycle and the time to primordium formation was lengthened. Strains S13-S20, obtained after 13-20 months of mycelial subculturing, also lacked the ability to produce fruiting bodies during cultivation experiments. Determination of reactive oxygen species (ROS) content as well as enzyme activity showed that decreased lignocellulase activity, along with excessive accumulation of ROS, was concomitant with the subculture-associated degeneration of V. volvacea. Reverse transcription polymerase chain reaction (RT-PCR) was eventually used to analyze the gene expression for lignocellulase and antioxidant enzymes in subcultured V. volvacea strains, with the results found to be consistent with prior observations regarding enzyme activities. These findings could form the basis of further studies on the degeneration mechanism of V. volvacea and other fungi.

Effects of single- and mixed-bacterial inoculation on the colonization and assembly of endophytic communities in plant roots.

The introduction and inoculation of beneficial bacteria in plants have consistently been considered as one of the most important ways to improve plant health and production. However, the effects of bacterial inoculation on the community assembly and composition of the root endophytic microbiome remain largely unknown. In this study, 55 strains were randomly isolated from tomato roots and then inoculated into wheat seeds singly or in combination. Most of the isolated bacterial strains showed an ability to produce lignocellulose-decomposing enzymes and promote plant growth. The results demonstrated that bacterial inoculation had a significant effect on the wheat root endophytic microbiome. The wheat root samples inoculated with single-bacterial species were significantly separated into two groups (A and B) that had different community structures and compositions. Among these, root endophytic communities for most wheat samples inoculated with a single-bacterial strain (Group A) were predominated by one or several bacterial species, mainly belonging to Enterobacterales. In contrast, only a few of the root samples inoculated with a single-bacterial strain (Group B) harbored a rich bacterial flora with relatively high bacterial diversity. However, wheat roots inoculated with a mixed bacterial complex were colonized by a more diverse and abundant bacterial flora, which was mainly composed of Enterobacterales, Actinomycetales, Bacillales, Pseudomonadales, and Rhizobiales. The results demonstrated that inoculation with bacterial complexes could help plants establish more balanced and beneficial endophytic communities. In most cases, bacterial inoculation does not result in successful colonization by the target bacterium in wheat roots. However, bacterial inoculation consistently had a significant effect on the root microbiome in plants. CAP analysis demonstrated that the variation in wheat root endophytic communities was significantly related to the taxonomic status and lignocellulose decomposition ability of the inoculated bacterial strain (p < 0.05). To reveal the role of lignocellulose degradation in shaping the root endophytic microbiome in wheat, four bacterial strains with different colonization abilities were selected for further transcriptome sequencing analysis. The results showed that, compared with that in the dominant bacterial species Ent_181 and Ent_189 of Group A, the expression of lignocellulose-decomposing enzymes was significantly downregulated in Bac_133 and Bac_71 (p < 0.05). In addition, we found that the dominant bacterial species of the tomato endophytic microbiome were more likely to become dominant populations in the wheat root microbiome. In general, our results demonstrated that lignocellulose-decomposing enzymes played a vital role in the formation of endophytes and their successful colonization of root tissues. This finding establishes a theoretical foundation for the development of broad-spectrum probiotics.

Tr-milRNA1 Contributes to Lignocellulase Secretion under Heat Stress by Regulating the Lectin-Type Cargo Receptor Gene Trvip36 in Trichoderma guizhouence NJAU 4742.

BACKGROUND: MicroRNA plays an important role in multifarious biological processes by regulating their corresponding target genes. However, the biological function and regulatory mechanism of fungal microRNA-like RNAs (milRNAs) remain poorly understood. METHODS: In this study, combined with deep sequencing and bioinformatics analysis, milRNAs and their targets from Trichoderma guizhouence NJAU 4742 were isolated and identified under solid-state fermentation (SSF) by using rice straw as the sole carbon source at 28 °C and 37 °C, respectively. RESULTS: A critical milRNA, TGA1_S04_31828 (Tr-milRNA1), was highly expressed under heat stress (37 °C) and adaptively regulated lignocellulase secretion. Overexpression of Tr-milRNA1 (OE-Tr-milRNA1) did not affect vegetative growth, but significantly increased lignocellulose utilization under heat stress. Based on the bioinformatics analysis and qPCR validation, a target of Tr-milRNA1 was identified as Trvip36, a lectin-type cargo receptor. The expression of Tr-milRNA1 and Trvip36 showed a divergent trend under SSF when the temperature was increased from 28 °C to 37 °C. In addition, the expression of Trvip36 was suppressed significantly in Tr-milRNA1 overexpression strain (OE-Tr-milRNA1). Compared with the wild type, deletion of Trvip36 (ΔTrvip36) significantly improved the secretion of lignocellulases by reducing the retention of lignocellulases in the ER under heat stress. CONCLUSIONS: Tr-milRNA1 from NJAU 4742 improved lignocellulose utilization under heat stress by regulating the expression of the corresponding target gene Trvip36. These findings might open avenues for exploring the mechanism of lignocellulase secretion in filamentous fungi.

Taxonomic Study of the Genus Pholiota (Strophariaceae, Basidiomycota) in Korea.

The genus Pholiota (Strophariaceae, Basidiomycota) is made up of wood-rotting saprotrophic mushrooms characterized by a yellow or brown pileus with scales and/or slimy, and by a brownish smooth spore with a germ pore. However, these features are not enough to distinguish its species, or separate the genus Pholiota from other brown-spored wood-rotting genera such as Hypholoma and Stropharia. Although internal transcribed spacer (ITS) sequence-based identification has improved identification accuracy for species of Pholiota, most Pholiota species in Korea are reported based on morphological features. To evaluate the taxonomy of Pholiota species, we investigated 62 specimens collected from 1999 to 2019 in Korea using ITS sequence analysis and morphological observation. Twelve of the 16 recorded Pholiota species in Korea were identified. While eight species were clearly separated, the ITS analysis did not distinguish three in the Pholiota adiposa complex. Therefore, further investigation is required to distinguish these three species. ITS sequences deposited in GenBank confirm that P. highlandensis exists in Korea. The presence of the other four Pholiota species could not be confirmed through specimens or sequence information in GenBank. A taxonomic key and the ITS sequence data for Korean Pholiota species are included and can be good baselines for further research on Pholiota taxonomy and diversity.

Improved Production of Majority Cellulases in Trichoderma reesei by Integration of cbh1 Gene From Chaetomium thermophilum.

Lignocellulose is an abundant waste resource and has been considered as a promising material for production of biofuels or other valuable bio-products. Currently, one of the major bottlenecks in the economic utilization of lignocellulosic materials is the cost-efficiency of converting lignocellulose into soluble sugars for fermentation. One way to address this problem is to seek superior lignocellulose degradation enzymes or further improve current production yields of lignocellulases. In the present study, the lignocellulose degradation capacity of a thermophilic fungus Chaetomium thermophilum was firstly evaluated and compared to that of the biotechnological workhorse Trichoderma reesei. The data demonstrated that compared to T. reesei, C. thermophilum displayed substantially higher cellulose-utilizing efficiency with relatively lower production of cellulases, indicating that better cellulases might exist in C. thermophilum. Comparison of the protein secretome between C. thermophilum and T. reesei showed that the secreted protein categories were quite different in these two species. In addition, to prove that cellulases in C. thermophilum had better enzymatic properties, the major cellulase cellobiohydrolase I (CBH1) from C. thermophilum and T. reesei were firstly characterized, respectively. The data showed that the specific activity of C. thermophilum CBH1 was about 4.5-fold higher than T. reesei CBH1 in a wide range of temperatures and pH. To explore whether increasing CBH1 activity in T. reesei could contribute to improving the overall cellulose-utilizing efficiency of T. reesei, T. reesei cbh1 gene was replaced with C. thermophilum cbh1 gene by integration of C. thermophilum cbh1 gene into T. reesei cbh1 gene locus. The data surprisingly showed that this gene replacement not only increased the cellobiohydrolase activities by around 4.1-fold, but also resulted in stronger induction of other cellulases genes, which caused the filter paper activities, Azo-CMC activities and ß-glucosidase activities increased by about 2.2, 1.9, and 2.3-fold, respectively. The study here not only provided new resources of superior cellulases genes and new strategy to improve the cellulase production in T. reesei, but also contribute to opening the path for fundamental research on C. thermophilum.

[Co-expression of lignocellulase from termite and their endosymbionts].

Natural lignocellulosic materials contain cellulose, hemicellulose, and lignin. Cellulose hydrolysis to glucose requires a series of lignocellulases. Recently, the research on the synergistic effect of lignocellulases has become a new research focus. Here, four lignocellulase genes encoding ß-glucosidase, endo-1,4-ß-glucanase, xylanase and laccase from termite and their endosymbionts were cloned into pETDuet-1 and pRSFDuet-1 and expressed in Escherichia coli. After SDS-PAGE analysis, the corresponding protein bands consistent with the theoretical values were observed and all the proteins showed enzyme activities. We used phosphoric acid swollen cellulose (PASC) as substrate to measure the synergistic effect of crude extracts of co-expressing enzymes and the mixture of single enzyme. The co-expressed enzymes increased the degradation efficiency of PASC by 44% compared with the single enzyme mixture; while the degradation rate increased by 34% and 20%, respectively when using filter paper and corn cob pretreated with phosphoric acid as substrates. The degradation efficiency of the co-expressed enzymes was higher than the total efficiency of the single enzyme mixture.

Co-expression of lignocellulase from termite and their endosymbionts / 生物工程学报

Natural lignocellulosic materials contain cellulose, hemicellulose, and lignin. Cellulose hydrolysis to glucose requires a series of lignocellulases. Recently, the research on the synergistic effect of lignocellulases has become a new research focus. Here, four lignocellulase genes encoding β-glucosidase, endo-1,4-β-glucanase, xylanase and laccase from termite and their endosymbionts were cloned into pETDuet-1 and pRSFDuet-1 and expressed in Escherichia coli. After SDS-PAGE analysis, the corresponding protein bands consistent with the theoretical values were observed and all the proteins showed enzyme activities. We used phosphoric acid swollen cellulose (PASC) as substrate to measure the synergistic effect of crude extracts of co-expressing enzymes and the mixture of single enzyme. The co-expressed enzymes increased the degradation efficiency of PASC by 44% compared with the single enzyme mixture; while the degradation rate increased by 34% and 20%, respectively when using filter paper and corn cob pretreated with phosphoric acid as substrates. The degradation efficiency of the co-expressed enzymes was higher than the total efficiency of the single enzyme mixture.

Isolation of fungi from dung of wild herbivores for application in bioethanol production

ABSTRACT Producing biofuels such as ethanol from non-food plant material has the potential to meet transportation fuel requirements in many African countries without impacting directly on food security. The current shortcomings in biomass processing are inefficient fermentation of plant sugars, such as xylose, especially at high temperatures, lack of fermenting microbes that are able to resist inhibitors associated with pre-treated plant material and lack of effective lignocellulolytic enzymes for complete hydrolysis of plant polysaccharides. Due to the presence of residual partially degraded lignocellulose in the gut, the dung of herbivores can be considered as a natural source of pre-treated lignocellulose. A total of 101 fungi were isolated (36 yeast and 65 mould isolates). Six yeast isolates produced ethanol during growth on xylose while three were able to grow at 42 °C. This is a desirable growth temperature as it is closer to that which is used during the cellulose hydrolysis process. From the yeast isolates, six isolates were able to tolerate 2 g/L acetic acid and one tolerated 2 g/L furfural in the growth media. These inhibitors are normally generated during the pre-treatment step. When grown on pre-treated thatch grass, Aspergillus species were dominant in secretion of endo-glucanase, xylanase and mannanase.

Isolation of fungi from dung of wild herbivores for application in bioethanol production.

Producing biofuels such as ethanol from non-food plant material has the potential to meet transportation fuel requirements in many African countries without impacting directly on food security. The current shortcomings in biomass processing are inefficient fermentation of plant sugars, such as xylose, especially at high temperatures, lack of fermenting microbes that are able to resist inhibitors associated with pre-treated plant material and lack of effective lignocellulolytic enzymes for complete hydrolysis of plant polysaccharides. Due to the presence of residual partially degraded lignocellulose in the gut, the dung of herbivores can be considered as a natural source of pre-treated lignocellulose. A total of 101 fungi were isolated (36 yeast and 65 mould isolates). Six yeast isolates produced ethanol during growth on xylose while three were able to grow at 42°C. This is a desirable growth temperature as it is closer to that which is used during the cellulose hydrolysis process. From the yeast isolates, six isolates were able to tolerate 2g/L acetic acid and one tolerated 2g/L furfural in the growth media. These inhibitors are normally generated during the pre-treatment step. When grown on pre-treated thatch grass, Aspergillus species were dominant in secretion of endo-glucanase, xylanase and mannanase.

Metatranscriptome of the protistan community in Reticulitermes flaviceps.

The hindgut of lower termites harbors various symbiotic protists, which perform varied functions in lignocellulose decomposition. As termites are social insects, the species and numbers of these flagellated protists in the termite gut vary among the different castes. Juvenile hormones (JHs) can regulate caste differentiation in termites. In this study, we used the juvenile hormone analog fenoxycarb to induce termite workers (Reticulitermes flaviceps) to differentiate into pre-soldiers. A metatranscriptomic investigation of the protistan community was then performed by 454 pyrosequencing. From a thorough analysis based on 597 312 generated reads, we found that the starch and sucrose metabolism pathway was the most abundant pathway across the metatranscriptome. The current study demonstrates that the metatranscriptome of the protistan community in termites contains an abundance of lignocellulase, which plays a vital role in termite nutrition.

Involvement of the adaptor protein 3 complex in lignocellulase secretion in Neurospora crassa revealed by comparative genomic screening.

BACKGROUND: Lignocellulase hypersecretion has been achieved in industrial fungal workhorses such as Trichoderma reesei, but the underlying mechanism associated with this process is not well understood. Although previous comparative genomic studies have revealed that the mutagenic T. reesei strain RUT-C30 harbors hundreds of mutations compared with its parental strain QM6a, how these mutations actually contribute to the hypersecretion phenotype remains to be elucidated. RESULTS: In this study, we systematically screened gene knockout (KO) mutants in the cellulolytic fungus Neurospora crassa, which contains orthologs of potentially defective T. reesei RUT-C30 mutated genes. Of the 86 deletion mutants screened in N. crassa, 12 exhibited lignocellulase production more than 25% higher than in the wild-type (WT) strain and 4 showed nearly 25% lower secretion. We observed that the deletion of Ncap3m (NCU03998), which encodes the µ subunit of the adaptor protein 3 (AP-3) complex in N. crassa, led to the most significant increase in lignocellulase secretion under both Avicel and xylan culture conditions. Moreover, strains lacking the ß subunit of the AP-3 complex, encoded by Ncap3b (NCU06569), had a similar phenotype to ΔNcap3m, suggesting that the AP-3 complex is involved in lignocellulase secretion in N. crassa. We also found that the transcriptional abundance of major lignocellulase genes in ΔNcap3m was maintained at a relatively higher level during the late stage of fermentation compared with the WT, which might add to the hypersecretion phenotype. Finally, we found that importation of the T. reesei ap3m ortholog Trap3m into ΔNcap3m can genetically restore secretion of lignocellulases to normal levels, which suggests that the effect of the AP-3 complex on lignocellulase secretion is conserved in cellulolytic ascomycetes. CONCLUSIONS: Using the model cellulolytic fungus N. crassa, we explored potential hypersecretion-related mutations in T. reesei strain RUT-C30. Through systematic genetic screening of 86 corresponding orthologous KO mutants in N. crassa, we identified several genes, particularly those encoding the AP-3 complex that contribute to lignocellulase secretion. These findings will be useful for strain improvement in future lignocellulase and biomass-based chemical production.

Genome-wide analysis of the endoplasmic reticulum stress response during lignocellulase production in Neurospora crassa.

BACKGROUND: Lignocellulolytic fungal cells suffer endoplasmic reticulum (ER) stress during lignocellulase synthesis; however, an understanding of this integrated process on a genome-wide scale remains poor. Here, we undertook a systematic investigation of this process in Neurospora crassa (N. crassa) using transcriptomic analysis coupled with genetic screens. RESULTS: A set of 766 genes was identified as the ER stress response targets (ESRTs) in N. crassa under cellulose utilization conditions. Among these, the expression of 223 and 186 genes showed dependence on IRE-1 and HAC-1, respectively. A total of 527 available mutants for ESRT genes were screened, 249 of which exhibited ER stress susceptibility, including 100 genes with unknown function. Disruption of ire-1 or hac-1 in N. crassa did not affect transcriptional induction of lignocellulase genes by cellulose but severely affected secretion of the corresponding enzymes. A global investigation of transcription factors (TFs) discovered three novel regulators (RES-1, RES-2, RRG-2) involved in lignocellulase secretion. Production of lignocellulases in Δres-1 increased by more than 30% in comparison to wild type (WT), while secretion decreased by nearly 30% in strains Δres-2 and Δrrg-2. Transcriptional profiling of the three TF mutants suggests they are deeply involved in lignocellulase secretion and ER stress response. CONCLUSIONS: Here, we determined the transcriptional scope of the ER stress response during lignocellulase synthesis in the model cellulolytic fungus N. crassa. Through genome-wide mutant screening and analysis, dozens of novel genes were discovered to be involved in the process. The findings of this work will be useful for strain improvement to facilitate lignocellulase and biomass-based chemical production.