Functional analysis of two mitogen-activated protein kinases involved in thermal resistance of the predatory mite Neoseiulus californicus (Acari: Phytoseiidae).

Predatory mites are important biological control agents used against phytophagous mites and small insects. They face various environmental pressures, especially fluctuating climate factors. Neoseiulus californicus, a commercially available phytoseiid mite, is adapted to a wide range of temperature conditions. We investigated the regulatory mechanisms governing the plastic response of N. californicus for coping with environmental temperature variations. The mitogen-activated protein kinase (MAPK) signaling pathway is a highly conserved pathway of cell signal transduction that responds to environmental stress. We isolated two MAPKK genes (NcMAPKK4 and NcMAPKK6) from N. californicus and studied their functions. Developmental stage-specific expression level analysis showed that in adults, particularly females, NcMAPKK4 and NcMAPKK6 levels were higher than in other developmental stages. The expression level analysis at extremely high and low temperature conditions demonstrated that NcMAPKK4 could be induced significantly by adverse thermal stresses, whereas NcMAPKK6 distinctly responded to heat shock, indicating their different roles in thermal stress responses. After silencing of NcMAPKK4, both heat and cold resistance decreased significantly, whereas NcMAPKK6 knockdown had a greater influence on heat resistance. Knockdown of NcMAPKKs also reduced the activities of antioxidant enzymes, suggesting the regulation of NcMAPKKs was closely related to the antioxidant process in oxidative stress caused by external stimuli. These results indicate an important role of NcMAPKKs in the response to thermal stress and provide insight into the MAPK cascade pathway in the environmental adaptation mechanisms of phytoseiid mites.

Glucanase-Induced Stipe Wall Extension Shows Distinct Differences from Chitinase-Induced Stipe Wall Extension of Coprinopsis cinerea.

This study reports that a high concentration of the endo-ß-1,3-glucanase ENG (200 µg ml-1) induced heat-inactivated stipe wall extension of Coprinopsis cinerea, whereas a high concentration of the extracellular ß-glucosidase BGL2 (1,000 µg ml-1) did not; however, in combination, low concentrations of ENG (25 µg ml-1) and BGL2 (260 µg ml-1) induced heat-inactivated stipe cell wall extension. In contrast to the previously reported chitinase-reconstituted stipe wall extension, ß-1,3-glucanase-reconstituted heat-inactivated stipe cell wall extension initially exhibited a fast extension rate that quickly decreased to zero after approximately 60 min; the stipe cell wall extension induced by a high concentration of ß-1,3-glucanase did not result in stipe breakage during measurement, and the inner surfaces of glucanase-reconstituted extended cell walls still remained as amorphous matrices that did not appear to have been damaged. These distinctive features of the ß-1,3-glucanase-reconstituted wall extension may be because chitin chains are cross-linked not only to the nonreducing termini of the side chains and the backbones of ß-1,6 branched ß-1,3-glucans but also to other polysaccharides. Remarkably, a low concentration of either the ß-1,3-glucanase ENG or of chitinase ChiE1 did not induce heat-inactivated stipe wall extension, but a combination of these two enzymes, each at a low concentration, showed stipe cell wall extension activity that exhibited a steady and continuous wall extension profile. Therefore, we concluded that the stipe cell wall extension is the result of the synergistic actions of glucanases and chitinases.IMPORTANCE We previously reported that the chitinase could induce stipe wall extension and was involved in stipe elongation growth of the mushroom Coprinopsis cinerea In this study, we explored that ß-1,3-glucanase also induced stipe cell wall extension. Interestingly, the extension profile and extended ultra-architecture of ß-1,3-glucanase-reconstituted stipe wall were different from those of chitinase-reconstituted stipe wall. However, ß-1,3-glucanase cooperated with chitinase to induce stipe cell wall extension. The significance of this synergy between glucanases and chitinases is that it enables a low concentration of active enzymes to induce wall extension, and the involvement of ß-1,3-glucanases is necessary for the cell wall remodeling and the addition of new ß-glucans during stipe elongation growth.

Chitinases Play a Key Role in Stipe Cell Wall Extension in the Mushroom Coprinopsis cinerea.

The elongation growth of the mushroom stipe is a characteristic but not well-understood morphogenetic event of basidiomycetes. We found that extending native stipe cell walls of Coprinopsis cinerea were associated with the release of N-acetylglucosamine and chitinbiose and with chitinase activity. Two chitinases among all detected chitinases from C. cinerea, ChiE1 and ChiIII, reconstituted heat-inactivated stipe wall extension and released N-acetylglucosamine and chitinbiose. Interestingly, both ChiE1 and ChiIII hydrolyze insoluble crystalline chitin powder, while other C. cinerea chitinases do not, suggesting that crystalline chitin components of the stipe cell wall are the target of action for ChiE1 and ChiIII. ChiE1- or ChiIII-reconstituted heat-inactivated stipe walls showed maximal extension activity at pH 4.5, consistent with the optimal pH for native stipe wall extension in vitro; ChiE1- or ChiIII-reconstituted heat-inactivated stipe wall extension activities were associated with stipe elongation growth regions; and the combination of ChiE1 and ChiIII showed a synergism to reconstitute heat-inactivated stipe wall extension at a low action concentration. Field emission scanning electron microscopy (FESEM) images showed that the inner surface of acid-induced extended native stipe cell walls and ChiE1- or ChiIII-reconstituted extended heat-inactivated stipe cell walls exhibited a partially broken parallel microfibril architecture; however, these broken transversely arranged microfibrils were not observed in the unextended stipe cell walls that were induced by neutral pH buffer or heat inactivation. Double knockdown of ChiE1 and ChiIII resulted in the reduction of stipe elongation, mycelium growth, and heat-sensitive cell wall extension of native stipes. These results indicate a chitinase-hydrolyzing mechanism for stipe cell wall extension.IMPORTANCE A remarkable feature in the development of basidiomycete fruiting bodies is stipe elongation growth that results primarily from manifold cell elongation. Some scientists have suggested that stipe elongation is the result of enzymatic hydrolysis of cell wall polysaccharides, while other scientists have proposed the possibility that stipe elongation results from nonhydrolytic disruption of the hydrogen bonds between cell wall polysaccharides. Here, we show direct evidence for a chitinase-hydrolyzing mechanism of stipe cell wall elongation in the model mushroom Coprinopsis cinerea that is different from the expansin nonhydrolysis mechanism of plant cell wall extension. We presumed that in the growing stipe cell walls, parallel chitin microfibrils are tethered by ß-1,6-branched ß-1,3-glucans, and that the breaking of the tether by chitinases leads to separation of these microfibrils to increase their spacing for insertion of new synthesized chitin and ß-1,3-glucans under turgor pressure in vivo.

Increased polysaccharide production and biosynthetic gene expressions in a submerged culture of Ganoderma lucidum by the overexpression of the homologous α-phosphoglucomutase gene.

This study aimed to improve the production of polysaccharide by engineering the biosynthetic pathway in Ganoderma lucidum through the overexpression of α-phosphoglucomutase (PGM) gene. PGM is responsible for the linkage between sugar catabolism and sugar anabolism. The effects of PGM gene overexpression on intracellular polysaccharide (IPS) content, extracellular polysaccharide (EPS) production and transcription levels of three genes encoding the enzymes involved in polysaccharide biosynthesis, including PGM, UDP-glucose pyrophosphorylase (UGP), and ß-1,3-glucan synthase (GLS), were investigated. The maximum IPS content and EPS production in G. lucidum overexpressing the PGM gene were 23.67 mg/100 mg dry weight and 1.76 g/L, respectively, which were higher by 40.5 and 44.3% than those of the wild-type strain. The transcription levels of PGM, UGP and GLS were upregulated by 4.77-, 1.51- and 1.53-fold, respectively, in the engineered strain, suggesting that increased polysaccharide biosynthesis may result from a higher expression of those genes.

Nitrile Hydratases: From Industrial Application to Acetamiprid and Thiacloprid Degradation.

The widespread application of neonicotinoid insecticides (NEOs) in agriculture causes a series of environmental and ecological problems. Microbial remediation is a popular approach to relieve these negative impacts, but the associated molecular mechanisms are rarely explored. Nitrile hydratase (NHase), an enzyme commonly used in industry for amide production, was discovered to be responsible for the degradation of acetamiprid (ACE) and thiacloprid (THI) by microbes. Since then, research into NHases in NEO degradation has attracted increasing attention. In this review, microbial degradation of ACE and THI is briefly described. We then focus on NHase evolution, gene composition, maturation mechanisms, expression, and biochemical properties with regard to application of NHases in NEO degradation for bioremediation.

Comparative study of ß-glucan-degrading enzymes from Coprinopsis cinerea for their capacities to induce stipe cell wall extension.

We previously reported endo-ß-1,3-glucanase ENG in combination with ß-glucosidase BGL2 at low concentration induced stipe cell wall extension. This study further explored ENG could be replaced by endo-ß-1,3(4)-glucanase ENG16A in combination with BGL2 to induce stipe cell wall extension; similarly, BGL2 could be replaced by ß-glucosidase BGL1 to cooperate with ENG to induce stipe cell wall extension. However, ENG could not be replaced by exo-ß-1,3-glucanase EXG in combination with BGL2 to induce stipe cell wall extension, although EXG alone released higher level of soluble sugars from the stipe cell walls during the reconstituted wall extension than that released from the stipe cell walls by a combination of ENG16A or ENG and BGL2 or BGL1, which was different from chitinase-mediated stipe cell wall extension. These results indicate endo-ß-1,3-glucanases loosen the stipe cell wall, whereas exo-ß-1,3-glucanases and ß-glucosidases play a synergistic role to maintain a low and efficient concentration of endo-ß-1,3-glucanases for stipe cell wall extension. Furthermore, ENG was expressed at a very high level in the matured pilei, in contrast, ENG16A was expressed at a very high level in the elongating apical stipe. Therefore, ENG16A might be involved in stipe elongation growth, while ENG might participate in autolysis of pilei.

A novel endo-ß-1,6-glucanase from the mushroom Coprinopsis cinerea and its application in studying of cross-linking of ß-1,6-glucan and the wall extensibility in stipe cell walls.

We previously reported that chitinases reconstituted heat-inactivated stipe cell wall extension in a steady and continuous extension profile by cleaving chitins cross-linked to various polysaccahrides, whereas, endo-ß-1,3-glucanases reconstituted heat-inactivated stipe wall extension in a profile of an initially fast extension and subsequent termination of extension due to its degradation of ß-1,3-glucan but not other polysaccharides such as ß-1,6-glucans cross-linked to chitins. Thus, a novel endo-ß-1,6-glucanase, GH30A, from Coprinopsis cinerea was cloned and characterized to study cross-linking of ß-1,6-glucan and wall extensibility in stipe walls. GH30A had higher activity and better thermophilicity than reported ß-1,6-glucanases. GH30A hydrolyzed pustulan having ß-1,6-linkages but not other polysaccharides without ß-1,6-linkages; GH30A did not cleave gentiobiose and single ß-1,6-linkage branches in laminarin from Laminaria digitata but cut consecutive ß-1,6-linkage branches in laminarin from Eisenia bicyclis. GH30A reconstituted heated-inactivated stipe cell wall extension with release of glucose and gentiobiose, indicating that ß-1,6-glucans were present and cross-linked to chitins in stipe walls, and cleaving ß-1,6-glucans cross-linked to chitins by GH30A led to wall loosening for extension. However, GH30A individually or in combination with endo-ß-1,3-glucanase reconstituted-stipe wall extension profile was similar to individual endo-ß-1,3-glucanase's, exploring that chitins were also cross-linked to other polysaccharides besides ß-1,3-glucans and ß-1,6-glucans.

A novel thermophilic exochitinase ChiEn3 from Coprinopsis cinerea exhibits a hyperhydrolytic activity toward 85% deacetylated chitosan and a significant application to preparation of chitooligosaccharides from the chitosan.

ChiEn3 from Coprinopsis cinerea was characterized as an exo-acting chitinase with a processivity. ChiEn3 hydrolyzed only soluble chitin and exhibited a hyperhydrolytic activity toward 85% deacetylated chitosan which was 33.6-fold higher than its hydrolytic activity toward glycol chitin. Its maximum hydrolytic activity was observed at 60 °C and retained 66.2% of hydrolytic activity after 60 min incubation at 60 °C. Commercial 85% deacetylated chitosan was degraded by ChiEn3 to a series of COSs with a DP of 2-20 in which COSs with a DP of 3-6 were dominant, whereas, lab-prepared chitosan (FA = 0.65) was degraded by ChiEn3 to COSs with a DP of 2-10 in which the AA dimer was dominant. DPPH-radical-scavenging activity of ChiEn3-digested products of 85% deacetylated chitosan was 3.32-fold higher than that of undigested 85% deacetylated chitosan. Therefore, ChiEn3 shows a valuable advantage for application to the preparation of COSs from commercial 85% deacetylated chitosan.

HPAEC-PAD and Q-TOF-MS/MS analysis reveal a novel mode of action of endo-ß-1,3(4)-d-glucanase Eng16A from coprinopsis cinerea on barley ß-glucan.

We previously reported that an endo-ß-1,3(4)-d-glucanase, Eng16A, from C. cinerea shows a higher degradation activity toward barley ß-glucan than laminarin. HPAEC-PAD and Q-TOF-MS/MS analyses show that Eng16A-digestion products of barley ß-glucan not only contain some oligosaccharides with (1 → 3)-ß-linkage adjacent to the reducing end, which is consistent with ß-1,3(4)-glucanase-digestion products, but also include some oligosaccharides containing (1 → 4)-ß-linkage adjacent to the reducing end which is consistent with cellulase-digestion products. Thus, Eng16A possesses both cellulase and ß-1,3(4)-glucanase activities. Because Eng16A does not degrade cellulose, we propose that the insertion of a (1 → 3)-ß-linkage among the groups of (1 → 4)-ß-linkages may make these (1 → 4)-ß-linkages prone to cleavage by Eng16A. Furthermore, Eng16A also possesses transglycosylation activity which leads to some products containing one or a few consecutive (1 → 3)-ß-linkages adjacent to the non-reducing end. Therefore, HPAEC-PAD and Q-TOF-MS/MS analyses provide an efficient approach to reveal complicated modes of action of some endo-ß-1,3(4)-d-glucanases on barley ß-glucan.

Improved Polysaccharide Production by Homologous Co-overexpression of Phosphoglucomutase and UDP Glucose Pyrophosphorylase Genes in the Mushroom Coprinopsis cinerea.

Coprinopsis polysaccharides exhibit hypoglycemic and antioxidant activities. In this report, increases in polysaccharide production by homologous co-overexpression or individual homologous overexpression of phosphoglucomutase and UDP glucose pyrophosphorylase gene in Coprinopsis cinerea, which participate in polysaccharide biosynthesis. The transcription levels of the target genes were upregulated significantly in the oePGM-UGP strain when compared with the oePGM or oeUGP strain. The maximum intracellular polysaccharide content obtained in the oePGM-UGP strain was 1.49-fold higher than that of the WT strain, whereas a slight improvement in polysaccharide production was obtained in the oePGM and oeUGP strains. Extracellular polysaccharide production was enhanced by 75% in the oePGM-UGP strain when compared with that of the WT strain, whereas improvements of 30% and 16% were observed for the oePGM and oeUGP strains, respectively. These results show that multiple interventions in polysaccharide biosynthesis pathways of Basidiomycetes might improve polysaccharide yields when compared with that of single interventions.

ChiE1 from Coprinopsis cinerea is Characterized as a Processive Exochitinase and Revealed to Have a Significant Synergistic Action with Endochitinase ChiIII on Chitin Degradation.

Fruiting bodies that exhibit strong autolysis of Coprinopsis cinerea are a good resource for the chitinolytic system. In this study, a new Chitinase ChiE1 from C. cinerea was cloned, heterologously expressed, and characterized. Biochemical analysis demonstrated that ChiE1 is an exochitinase with a processive mode of action. Although ChiE1 contains only a single catalytic domain without a binding domain, it can bind to and degrade insoluble chitin powder and colloidal chitin. The combination of ChiE1 and C. cinerea endochitinase ChiIII could increase the amount of reducing sugar released from chitin powder by approximately 120% compared to using ChiE1 and ChiIII alone. The synergistic action of ChiE1 and ChiIII on degradation of chitin powder is higher than all previously reported synergism of chitinases. The recombinant Chitinase ChiE1 expressed in Pichia pastoris may be used as a synergistic chitinase for a reconstituted chitinolytic system for agricultural, biological, and environmental applications.

Heterologous Expression and Characterization of a Novel Chitinase (ChiEn1) from Coprinopsis cinerea and its Synergism in the Degradation of Chitin.

Chitinase ChiEn1 did not hydrolyze insoluble chitin but showed hydrolysis and transglycosylation activities toward chitin-oligosaccharides. Interestingly, the addition of ChiEn1 increased the amount of reducing sugars released from chitin powder by endochitinase ChiIII by 105.0%, and among the released reducing sugars the amount of (GlcNAc)2 was increased by 149.5%, whereas the amount of GlcNAc was decreased by 10.3%. The percentage of GlcNAc in the products of chitin powder with the combined ChiIII and ChiEn1 was close to that in the products of chitin-oligosaccharides with ChiEn1, rather than that with ChiIII. These results indicate that chitin polymers are first degraded into chitin oligosaccharides by ChiIII and the latter are further degraded to monomers and dimers by ChiEn1, and the synergistic action of ChiEn1 and ChiIII is involved in the efficient degradation of chitin in cell walls during pileus autolysis. The structure modeling explores the molecular base of ChiEn1 action.

The study of fingerprint characteristics of Dayi Pu-Erh tea using a fully automatic HS-SPME/GC-MS and combined chemometrics method.

The quality of tea is presently evaluated by the sensory assessment of professional tea tasters, however, this approach is both inconsistent and inaccurate. A more standardized and efficient method is urgently needed to objectively evaluate tea quality. In this study, the chemical fingerprint of 7 different Dayi Pu-erh tea brands and 3 different Ya'an tea brands on the market were analyzed using fully automatic headspace solid-phase microextraction (HS-SPME) combined with gas chromatography-mass spectrometry (GC-MS). A total of 78 volatiles were separated, among 75 volatiles were identified by GC-MS in seven Dayi Pu-erh teas, and the major chemical components included methoxyphenolic compounds, hydrocarbons, and alcohol compounds, such as 1,2,3-trimethoxybenzene, 1,2,4-trimethoxybenzene, 2,6,10,14-tetramethyl-pentadecane, linalool and its oxides, α-terpineol, and phytol. The overlapping ratio of peaks (ORP) of the chromatogram in the seven Dayi Pu-erh tea samples was greater than 89.55%, whereas the ORP of Ya'an tea samples was less than 79.10%. The similarity and differences of the Dayi Pu-erh tea samples were also characterized using correlation coefficient similarity and principal component analysis (PCA). The results showed that the correlation coefficient of similarity of the seven Dayi Pu-erh tea samples was greater than 0.820 and was gathered in a specific area, which showed that samples from different brands were basically the same, despite have some slightly differences of chemical indexes was found. These results showed that the GC-MS fingerprint combined with the PCA approach can be used as an effective tool for the quality assessment and control of Pu-erh tea.