Proteomic Characterization of Lignocellulolytic Enzymes Secreted by the Insect-Associated Fungus
            <i>Daldinia decipiens</i>
            oita, Isolated from a Forest in Northern Japan

Hori, Chiaki; Song, Ruopu; Matsumoto, Kazuki; Matsumoto, Ruy; Minkoff, Benjamin B.; Oita, Shuzo; Hara, Hideho; Takasuka, Taichi E.

doi:10.1128/aem.02350-19

Cited by 15 publications

(8 citation statements)

References 48 publications

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“…These promoters are induced particularly in the presence of soluble oligosaccharides produced from xylan and cellulose. Similar results of the inducible promoter were reported in the case of the well-known fungus Daldinia decipiens oita and T. reesei when grown on different carbon sources ( Novy et al, 2019 ; Hori et al, 2020 ). Surprisingly, the genomic analysis showed that Daldinia decipiens oita lacks the lignin-degrading enzymes ( Hori et al, 2020 ).…”

Section: Resultssupporting

confidence: 82%

Thermophilic Geobacillus WSUCF1 Secretome for Saccharification of Ammonia Fiber Expansion and Extractive Ammonia Pretreated Corn Stover

et al. 2022

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A thermophilic Geobacillus bacterial strain, WSUCF1 contains different carbohydrate-active enzymes (CAZymes) capable of hydrolyzing hemicellulose in lignocellulosic biomass. We used proteomic, genomic, and bioinformatic tools, and genomic data to analyze the relative abundance of cellulolytic, hemicellulolytic, and lignin modifying enzymes present in the secretomes. Results showed that CAZyme profiles of secretomes varied based on the substrate type and complexity, composition, and pretreatment conditions. The enzyme activity of secretomes also changed depending on the substrate used. The secretomes were used in combination with commercial and purified enzymes to carry out saccharification of ammonia fiber expansion (AFEX)-pretreated corn stover and extractive ammonia (EA)-pretreated corn stover. When WSUCF1 bacterial secretome produced at different conditions was combined with a small percentage of commercial enzymes, we observed efficient saccharification of EA-CS, and the results were comparable to using a commercial enzyme cocktail (87% glucan and 70% xylan conversion). It also opens the possibility of producing CAZymes in a biorefinery using inexpensive substrates, such as AFEX-pretreated corn stover and Avicel, and eliminates expensive enzyme processing steps that are used in enzyme manufacturing. Implementing in-house enzyme production is expected to significantly reduce the cost of enzymes and biofuel processing cost.

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Section: Resultssupporting

confidence: 82%

Thermophilic Geobacillus WSUCF1 Secretome for Saccharification of Ammonia Fiber Expansion and Extractive Ammonia Pretreated Corn Stover

et al. 2022

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“…The sample preparation and proteomic analysis were performed by following the previous study [54]. Briefly, 10 μg equivalent of the wheat germ cell-free extract (WEPRO7240H Lot: 15AQ02, CellFree Sciences) was precipitated by 20% (vol/vol) trichloroacetate (Wako Pure Chemical Corporation), and incubated for 20 min on ice, followed by ice-cold acetone wash for two times.…”

Section: Proteomic Analysis Of the Wheat Germ Cell-free Extractmentioning

confidence: 99%

Reconstitution of Drosophila and human chromatins by wheat germ cell-free co-expression system

et al. 2020

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Background Elaboration of the epigenetic regulation of chromatin is a long-standing aim in molecular and cellular biology. Hence, there is a great demand for the development of in vitro methods to reconstitute chromatin that can be used directly for biochemical assays. The widely used wheat germ cell-free protein expression method provides broad applications to investigate the function and structure of eukaryotic proteins. Such advantages, including high translation efficiency, flexibility, and possible automatization, are beneficial for achieving native-like chromatin substrates for in vitro studies. Results We describe a novel, single-step in vitro chromatin assembly method by using the wheat germ cell-free protein synthesis. We demonstrated that both Drosophila and human chromatins can be reconstituted in the course of the in vitro translation of core histones by the addition of chromatin assembly factors, circular plasmid, and topoisomerase I in an ATP-dependent manner. Drosophila chromatin assembly was performed in 4 h at 26 °C, in the presence of premixed mRNAs encoding the core histones, dAcf1/dISWI chromatin remodeling complex, and nucleosome assembly protein, dNAP1. Similarly, the human chromatin was assembled by co-expressing the human core histones with Drosophila chromatin remodeling factor, dISWI, and chromatin chaperone, dNLP, for 6 h at 26 °C. The presence of reconstituted chromatin was monitored by DNA supercoiling assay, also the regular spacing of nucleosomes was assessed by Micrococcal nuclease assay. Furthermore, Drosophila linker histone H1-containing chromatin was reconstituted, affirming that the in vitro assembled chromatin is suitable for downstream applications. Conclusions The method described in this study allows the assembly of Drosophila and human chromatins, possibly in native-like form, by using a wheat germ cell-free protein expression. Although both chromatins were reconstituted successfully, there were unexpected differences with respect to the required ratio of histone-coding mRNAs and the reaction time. Overall, our new in vitro chromatin reconstitution method will aid to characterize the unrevealed structure, function, and regulation of chromatin dynamics.

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“…S2), which lets cells grow and secrete a basal level of CAZymes that could subsequently hydrolyze LBG, including b-mannanase (GH5; SACTE_2347) (8,13). Alternatively, in a natural symbiont environment, a series of oligosaccharides such as mannooligosaccharides would be provided by symbiont microbes that can decompose plant cell wall components (27). When cells are exposed to mannose or mannobiose, they are imported into the cell by a putative mannose transporter (SACTE_5236, SACTE_5237, and SACTE_5238) (28), and this displaces SsManR from the 4 promoter regions (Prom_0383, Prom_0504, Prom_5235, and Prom_6282).…”

Section: Discussionmentioning

confidence: 99%

Mannose- and Mannobiose-Specific Responses of the Insect-Associated Cellulolytic Bacterium Streptomyces sp. Strain SirexAA-E

Ohashi

Hataya

Nakata

et al. 2021

Appl Environ Microbiol

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The cellulolytic insect symbiont bacterium, Streptomyces sp. SirexAA-E (SirexAA-E) secretes a suite of Carbohydrate Active enZymes (CAZymes), which are involved in the degradation of various polysaccharides in the plant cell wall, in response to the available carbon sources. Here, we examined a poorly understood response of this bacterium to mannan, one of the major plant cell wall components. SirexAA-E grew well on mannose, carboxymethyl cellulose (CMC), and locust bean gum (LBG) as sole carbon sources in the culture medium. The secreted proteins from each culture supernatant were tested for their polysaccharide-degrading ability, and the composition of secreted CAZymes in each sample was determined by LC-MS/MS. The results indicated that mannose, LBG, and CMC induced the secretion of mannan and cellulose-degrading enzymes. Interestingly, two α-1,2-mannosidases were abundantly secreted during growth on mannose and LBG. By genomic analysis, we found a unique 12 bp palindromic sequence motif at 4 locations in the SirexAA-E genome, two of which were found upstream of the above-mentioned α-1,2-mannosidase genes, along with a newly identified mannose and mannobiose-responsive transcriptional regulator, SsManR. Furthermore, the previously reported cellobiose-responsive repressor, SsCebR, was determined to also use mannobiose as an effector ligand. To test whether mannobiose induces the sets of genes under the control of the two regulators, SirexAA-E was grown on mannobiose, and the secretome composition was analyzed. As hypothesized, the composition of the mannobiose secretome combined sets of CAZymes found in both LBG and CMC secretomes, and so are likely under the regulation of both SsManR and SsCebR. Importance Streptomyces sp. SirexAA-E, a microbial symbiont of biomass harvesting insects, secretes a suite of polysaccharide-degrading enzymes dependent on the available carbon sources. However, the response of this bacterium to mannan has not been documented. In this study, we investigated the response of this bacterium to mannose, mannobiose, and galactomannan (LBG). By combining biochemical, proteomic, and genomic approaches, we discovered a novel mannose and mannobiose responsive transcriptional regulator, SsManR, which selectively regulates three α-1,2-mannosidase-coding genes. We also demonstrated that the previously described cellobiose responsive regulator, SsCebR, could use mannobiose as an effector ligand. Overall, our findings suggest that the Streptomyces sp. SirexAA-E responds to mannose and mannooligosaccharides through two different transcriptional repressors that regulate the secretion of the plant cell wall-degrading enzymes to extract carbon sources in the host environment.

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Proteomic Characterization of Lignocellulolytic Enzymes Secreted by the Insect-Associated Fungus Daldinia decipiens oita, Isolated from a Forest in Northern Japan

Cited by 15 publications

References 48 publications

Thermophilic Geobacillus WSUCF1 Secretome for Saccharification of Ammonia Fiber Expansion and Extractive Ammonia Pretreated Corn Stover

Thermophilic Geobacillus WSUCF1 Secretome for Saccharification of Ammonia Fiber Expansion and Extractive Ammonia Pretreated Corn Stover

Reconstitution of Drosophila and human chromatins by wheat germ cell-free co-expression system

Mannose- and Mannobiose-Specific Responses of the Insect-Associated Cellulolytic Bacterium Streptomyces sp. Strain SirexAA-E

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