Liquid-Solid Interfaces under Dynamic Shear Flow: Recent Insights into the Interfacial Slip.

The development of micro/nanofluidic techniques has recently revived interest in dynamic shear flow at liquid-solid interfaces. When the nature of the liquid-solid boundaries was revisited, the slip of the fluids relative to the solid wall was predicted theoretically and confirmed experimentally. This indicates that the molecular-level structures of the liquid-solid interfaces will be influenced by the liquid flow over certain temporal and spatial criteria. However, the fluid flow at the boundary layer still cannot be precisely predicted and effectively controlled, somehow limiting its practical applications. Here, we summarize the recent advances for the microscopic structures at the liquid-solid interfaces upon shear flow. Special attention was given to a second-order nonlinear optical technique, sum frequency generation vibrational spectroscopy, which is a powerful tool for exploring the molecular-level structures and structural dynamics at the liquid-solid interfaces and offering new insights into the molecular mechanisms of the fluid slip at the interfaces. Moreover, we discuss the possible approaches for controlling the interfacial slip at the molecular level and highlight the current challenges and opportunities. Although the theoretical framework of the slip at the liquid-solid interfaces is still incomplete, we hope that this Perspective will complement and enhance our understanding of various interfacial properties and phenomena with respect to practical non-equilibrium dynamic processes happening at the interfaces.

Transmembrane transport process and endoplasmic reticulum function facilitate the role of gene cel1b in cellulase production of Trichoderma reesei.

BACKGROUND: A total of 11 ß-glucosidases are predicted in the genome of Trichoderma reesei, which are of great importance for regulating cellulase biosynthesis. Nevertheless, the relevant function and regulation mechanism of each ß-glucosidase remained unknown. RESULTS: We evidenced that overexpression of cel1b dramatically decreased cellulase synthesis in T. reesei RUT-C30 both at the protein level and the mRNA level. In contrast, the deletion of cel1b did not noticeably affect cellulase production. Protein CEL1B was identified to be intracellular, being located in vacuole and cell membrane. The overexpression of cel1b reduced the intracellular pNPGase activity and intracellular/extracellular glucose concentration without inducing carbon catabolite repression. On the other hand, RNA-sequencing analysis showed the transmembrane transport process and endoplasmic reticulum function were affected noticeably by overexpressing cel1b. In particular, some important sugar transporters were notably downregulated, leading to a compromised cellular uptake of sugars including glucose and cellobiose. CONCLUSIONS: Our data suggests that the cellulase inhibition by cel1b overexpression was not due to the ß-glucosidase activity, but probably the dysfunction of the cellular transport process (particularly sugar transport) and endoplasmic reticulum (ER). These findings advance the knowledge of regulation mechanism of cellulase synthesis in filamentous fungi, which is the basis for rationally engineering T. reesei strains to improve cellulase production in industry.

Carbon dots for multicolor cell imaging and ultra-sensitive detection of multiple ions in living cells: One Stone for multiple Birds.

Given the significant impact of ions on environment pollution and human health, it is urgently needed to establish effective and convenient ion detection approaches, particularly in living cells. In this paper, we constructed multicolor N-doped-carbon dots (mPD-CDs) by facile one-step hydrothermal carbonization of m-phenylenediamine (mPD). mPD-CDs were successfully deployed for multicolor cellular imaging for animal cells, fungi, and bacteria in a wash-free way with high photostability and satisfactory biocompability. Moreover, mPD-CDs can be used as a fluorescent sensing probe for ultrasensitive detection of both iodide ion (I-) and typical heavy metals such as cadmium (Cd2+), copper (Cu2+), mercury (Hg2+), gadolinium (Gd3+), ferrous ion (Fe2+), Zinc (Zn2+), and ferric ion (Fe3+). This is the first report using CDs as optical sensing probe for the detection of Gd3+, and for detection of Fe3+ with fluorescence "turn on". More significantly, with these versatile and fascinating properties, we applied mPD-CDs for intracellular ion detection in living cells like Hep G2 and S. cerevisiae, and zebra fish. Altogether, mPD-CDs displayed great potential for multicolor cell imaging and the multiple ion detection in vitro and in vivo, presenting a promising strategy for in-situ ultrasensitive sensing of multiple metal ions in the environment and the biological systems.

Beneficial factors for biomineralization by ureolytic bacterium Sporosarcina pasteurii.

BACKGROUND: The ureolytic bacterium Sporosarcina pasteurii is well-known for its capability of microbially induced calcite precipitation (MICP), representing a great potential in constructional engineering and material applications. However, the molecular mechanism for its biomineralization remains unresolved, as few studies were carried out. RESULTS: The addition of urea into the culture medium provided an alkaline environment that is suitable for S. pasteurii. As compared to S. pasteurii cultivated without urea, S. pasteurii grown with urea showed faster growth and urease production, better shape, more negative surface charge and higher biomineralization ability. To survive the unfavorable growth environment due to the absence of urea, S. pasteurii up-regulated the expression of genes involved in urease production, ATPase synthesis and flagella, possibly occupying resources that can be deployed for MICP. As compared to non-mineralizing bacteria, S. pasteurii exhibited more negative cell surface charge for binding calcium ions and more robust cell structure as nucleation sites. During MICP process, the genes for ATPase synthesis in S. pasteurii was up-regulated while genes for urease production were unchanged. Interestingly, genes involved in flagella were down-regulated during MICP, which might lead to poor mobility of S. pasteurii. Meanwhile, genes in fatty acid degradation pathway were inhibited to maintain the intact cell structure found in calcite precipitation. Both weak mobility and intact cell structure are advantageous for S. pasteurii to serve as nucleation sites during MICP. CONCLUSIONS: Four factors are demonstrated to benefit the super performance of S. pasteurii in MICP. First, the good correlation of biomass growth and urease production of S. pasteurii provides sufficient biomass and urease simultaneously for improved biomineralization. Second, the highly negative cell surface charge of S. pasteurii is good for binding calcium ions. Third, the robust cell structure and fourth, the weak mobility, are key for S. pasteurii to be nucleation sites during MICP.

Comparative transcriptomic analysis reveals the significant pleiotropic regulatory effects of LmbU on lincomycin biosynthesis.

BACKGROUND: Lincomycin, produced by Streptomyces lincolnensis, is a lincosamide antibiotic and widely used for the treatment of the infective diseases caused by Gram-positive bacteria. The mechanisms of lincomycin biosynthesis have been deeply explored in recent years. However, the regulatory effects of LmbU that is a transcriptional regulator in lincomycin biosynthetic (lmb) gene cluster have not been fully addressed. RESULTS: LmbU was used to search for homologous LmbU (LmbU-like) proteins in the genomes of actinobacteria, and the results showed that LmbU-like proteins are highly distributed regulators in the biosynthetic gene clusters (BGCs) of secondary metabolites or/and out of the BGCs in actinomycetes. The overexpression, inactivation and complementation of the lmbU gene indicated that LmbU positively controls lincomycin biosynthesis in S. lincolnensis. Comparative transcriptomic analysis further revealed that LmbU activates the 28 lmb genes at whole lmb cluster manner. Furthermore, LmbU represses the transcription of the non-lmb gene hpdA in the biosynthesis of L-tyrosine, the precursor of lincomycin. LmbU up-regulates nineteen non-lmb genes, which would be involved in multi-drug flux to self-resistance, nitrate and sugar transmembrane transport and utilization, and redox metabolisms. CONCLUSIONS: LmbU is a significant pleiotropic transcriptional regulator in lincomycin biosynthesis by entirely activating the lmb cluster and regulating the non-lmb genes in Streptomyces lincolnensis. Our results first revealed the pleiotropic regulatory function of LmbU, and shed new light on the transcriptional effects of LmbU-like family proteins on antibiotic biosynthesis in actinomycetes.

Characterization of three pathway-specific regulators for high production of monensin in Streptomyces cinnamonensis.

Monensin, a polyether ionophore antibiotic, is produced by Streptomyces cinnamonensis and worldwide used as a coccidiostat and growth-promoting agent in the field of animal feeding. The monensin biosynthetic gene cluster (mon) has been reported. In this study, the potential functions of three putatively pathway-specific regulators (MonH, MonRI, and MonRII) were clarified. The results from gene inactivation, complementation, and overexpression showed that MonH, MonRI, and MonRII positively regulate monensin production. Both MonH and MonRI are essential for monensin biosynthesis, while MonRII is non-essential and could be completely replaced by additional expression of monRI. Transcriptional analysis of the mon cluster by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and electrophoresis mobility shift assays (EMSAs) revealed a co-regulatory cascade process. MonH upregulates the transcription of monRII, and MonRII in turn enhances the transcription of monRI. MonRII is an autorepressor, while MonRI is an autoactivator. MonH activates the transcription of monCII-monE, and upregulates the transcription of monT that is repressed by MonRII. monAX and monD are activated by MonRI, and upregulated by MonRII. Co-regulation of those post-polyketide synthase (post-PKS) genes by MonH, MonRI, and MonRII would contribute to high production of monensin. These results shed new light on the transcriptional regulatory cascades of antibiotic biosynthesis in Streptomyces.

Highly efficient production of rhamnolipid in P. putida using a novel sacB-based system and mixed carbon source.

Pseudomonas putida KT2440, a GRAS strain, has been used for synthesizing bulk and fine chemicals. However, the gene editing tool to metabolically engineer KT2440 showed low efficiency. In this study, a novel sacB-based system pK51mobsacB was established to improve the efficiency for marker-free gene disruption. Then the rhamnolipid synthetic pathway was introduced in KT2440 and genes of the competitive pathways were deleted to lower the metabolic burden based on pK51mobsacB. A series of endogenous and synthetic promoters were used for fine tuning rhlAB expression. The limited supply of dTDP-L-rhamnose was enhanced by heterologous rmlBDAC expression. Cell growth and rhamnolipid production were well balanced by using glucose/glycerol as mixed carbon sources. The final strain produced 3.64 g/L at shake-flask and 19.77 g/L rhamnolipid in a 5 L fermenter, the highest obtained among metabolically engineered KT2440, which implied the potential of KT2440 as a promising microbial cell factory for industrial rhamnolipid production.

Intracellular Sugar Transporters Facilitate Cellulase Synthesis in Trichoderma reesei Using Lactose.

Sugar transporters play an important role in the cellulase production of lignocellulose-degrading fungi. Nevertheless, the role and function of these transporters are still unclear. Here we first report intracellular sugar transporters assisting cellulase production in Trichoderma reesei (T. reesei) using lactose. The mRNA levels of sugar transporter genes mfs, gst, and lac1 were substantially upregulated in T. reesei cultivated on lactose, with the most abundant mRNA levels at 24 h as compared to glucose. Moreover, the individual deletion of these sugar transporters significantly inhibited cellulase production, solid cell growth, and sporulation of T. reesei, suggesting they play a supporting role in cellulase production when grown in lactose. Surprisingly, MFS, GST, and LAC1 were mainly localized in the cytoplasm, with MFS and LAC1 in the endoplasmic reticulum (ER), representing the first discovery of intracellular sugar transporters involved in cellulase biosynthesis in lactose culture. The absence of the gene lac1 noticeably inhibited most of the crucial genes related to cellulase production, including cellulase-encoding genes, transcription factors, and sugar transporters, at 24 h, which was fully relieved at 48 h or 72 h, indicating that lac1 affects cellulase production more at the early step. This research advances the understanding of the function of intracellular sugar transporters in fungi, particularly for fungal cellulase production.

Demonstrating the Interfacial Polymer Thermal Transition from Coil-to-Globule to Coil-to-Stretch under Shear Flow Using SFG and MD Simulation.

Revealing interfacial shear-induced structural responsiveness has long been an important topic in that most fluids in nature and human life are in motion and cause interesting boundary phenomena. It is amazing how the polymer chain conformation or local structural features at a boundary change under the effective shear condition. In this study, microfluidic-assisted sum frequency generation (SFG) vibrational spectroscopy and all-atom molecular dynamics (MD) simulation are combined to reveal that the shear flow can effectively block the so-called thermal coil-to-globule transition of the poly(N-isopropylacrylamide) (PNIPAM) brushes on the solid substrate, and the normal coil-to-globule transition transfers to a coil-to-stretch one under shear flow with increasing ambient temperature. Such findings are attributed to the balance between the shear flow and the molecular interaction with respect to the polymer chains and adjacent water molecules, thus demonstrating the significant effect of the shear flow on the structural and dynamic behaviors of the polymer chains at the boundaries from the molecular level.

Discovery of ER-localized sugar transporters for cellulase production with lac1 being essential.

BACKGROUND: In the process of cellulose hydrolysis, carbohydrate hydrolysates are transported into cells through membrane transporters, and then affect the expression of cellulase-encoding genes. Sugar transporters play a crucial role in cellulase production in lignocellulolytic fungi, of which relatively few have been functionally validated to date and are all reported to be on cell membrane. RESULT: Through transcriptome analysis and qRT-PCR, three putative MFS sugar transporters GST, MFS, and LAC1 were found to display significantly higher mRNA levels in T. reesei grown on cellulose than on glucose. The individual deletion of these three genes compromised cellulase production and delayed sugar absorption by 24 h in T. reesei. Nevertheless, they transported pretty low level of sugars, including galactose, lactose, and mannose, and did not transport glucose, when expressed in yeast system. Meanwhile, all three transporters were unexpectedly found to be intracellular, being located in endoplasmic reticulum (ER). Particularly, the knockout of lac1 almost abolished cellulase production, and significantly inhibited biomass generation regardless of sugar types, indicating that lac1 is essential for cellulase production and biomass formation. The absence of lac1 upregulated genes involved in ribosome biogenesis, while downregulated genes in cellulase production, protein processing in ER (particularly protein glycosylation), and lipid biosynthesis. The inhibition of lac1 deletion on the transcriptional levels of genes related to cellulase biosynthesis was restored after 72 h, but the cellulase production was still inhibited, indicating lac1 might pose a post-transcription regulation on cellulase production that are independent on the known cellulase regulation mediated by CRT1 and XYR1. CONCLUSION: For the first time, intracellular sugar transporters (mfs, gst, and lac1) facilitating cellulase production were identified, which was distributed in ER. Their sugar transporting ability was very weak, indicating that they might be related to sugar utilization inside cells rather than the cellular sugar uptake. More importantly, sugar transporter lac1 is first found to be essential for cellulase production and biomass formation by affecting protein processing in ER (particularly protein glycosylation) and lipid biosynthesis. The effect of LAC1 on cellulase production seems to be post-transcriptional at late stage of cellulase production, independent on the well-known cellulase regulation mediated by CRT1 and XYR1. These findings improve the understanding of intracellular sugar transporters in fungi and their important role in cellulase synthesis.

Intron retention coupled with nonsense-mediated decay is involved in cellulase biosynthesis in cellulolytic fungi.

BACKGROUND: Knowledge on regulatory networks associated with cellulase biosynthesis is prerequisite for exploitation of such regulatory systems in enhancing cellulase production with low cost. The biological functions of intron retention (IR) and nonsense-mediated mRNA decay (NMD) in filamentous fungi is lack of study, let alone their roles in cellulase biosynthesis. RESULTS: We found that major cellulase genes (cel7a, cel7b, and cel3a) exhibited concomitant decrease in IR rates and increase in their gene expression in T. reesei under cellulase-producing condition (cellulose and lactose) that was accompanied with a more active NMD pathway, as compared to cellulase non-producing condition (glucose). In the presence of the NMD pathway inhibitor that successfully repressed the NMD pathway, the mRNA levels of cellulase genes were sharply down-regulated, but the rates of IR in these genes were significantly up-regulated. Consistently, the cellulase activities were severely inhibited. In addition, the NMD pathway inhibitor caused the downregulated mRNA levels of two important genes of the target of rapamycin (TOR) pathway, trfkbp12 and trTOR1. The absence of gene trfkbp12 made the cellulase production in T. reesei more sensitive to the NMD pathway inhibitor. CONCLUSIONS: All these findings suggest that the IR of cellulase genes regulates their own gene expression by coupling with the NMD pathway, which might involve the TOR pathway. Our results provide better understanding on intron retention, the NMD pathway, and cellulase production mechanism in filamentous fungi.

Plant-derived Ca, N, S-Doped carbon dots for fast universal cell imaging and intracellular Congo red detection.

Congo red (CR) is a hazardous pigment, posing increasing dangerous to the environment and human health. However, the in-situ detection of CR in living cells has not been reported, as far as we know. Here, negatively-charged green-emitting Ca, N, S-doped carbon dots (Mis-mPD-CDs) were fabricated from plant and m-phenylenediamine (mPD) by facile one-step hydrothermal carbonization. Mis-mPD-CDs were capable of rapidly detecting CR on the basis of their fluorescence quenching by CR due to the inner filter effect. This CR detection based on Mis-mPD-CDs displayed a linear range of 0.2-1.2 µM and a low limit of detection (58 nM), and was not interfered by metal ions, important biological molecules, and other dyes, showing high sensitivity and selectivity. More interestingly, Mis-mPD-CDs can rapidly enter and label animal cells (A549, 4T1, and HUVEC), fungi (S. cerevisiae, C. albicans, and T. reesei), and bacteria (E. coli and S. aureus) for long term with high stability and appealing biocompatibility. Based on these compelling characteristics, we applied Mis-mPD-CDs for sensing and imaging CR in living cells (A549, C. albicans, E. coli, and S. aureus) and zebra fish. On the other hand, the quantitative detection of CR by Mis-mPD-CDs was realized in real samples like fish tissues and industrial wastewater. This is the first report on applying CDs for rapid CR detection in living cells and in vivo. Mis-mPD-CDs provides a novel efficient platform for probing intracellular CR, expanding the applications of CDs as biosensors for toxic dyes.

Chitosan-modified fluorescent dye for simple, fast, and in-situ measurement of fungal cell growth in the presence of insoluble compounds.

The measurement of fungal cell growth in submerged culture systems containing insoluble compounds is essential yet difficult due to the interferences from the insoluble compounds like biopolymers. Here, we developed a fluorescent strategy based on chitosan-modified fluorescein isothiocyanate (GC-FITC) to monitor the cell growth of lignocellulosic fungi cultivated on biopolymers. GC-FITC could stain only lignocellulosic fungi (Tricoderma reesei, Penicillium oxalicum, Aspergillus nidulans, and Neurospora crassa), but not biopolymers (cellulose, xylan, pectin, or lignin), excluding the interferences from these insoluble biopolymer. Moreover, a linear relationship was observed between the fluorescence intensity of GC-FITC absorbed by lignocellulosic fungi and the biomass of lignocellulosic fungi. Therefore, GC-FITC was leveraged to monitor the cell growth of lignocellulosic fungi when using biopolymers like cellulose as the carbon sources, which is faster, more convenient, time-saving, and cost-effective than the existing methods using protein/DNA content measurement. GC-FITC offers a powerful tool to detect fungal growth in culture systems with insoluble materials.

High-dose rapamycin exerts a temporary impact on T. reesei RUT-C30 through gene trFKBP12.

BACKGROUND: Knowledge with respect to regulatory systems for cellulase production is prerequisite for exploitation of such regulatory networks to increase cellulase production, improve fermentation efficiency and reduce the relevant production cost. The target of rapamycin (TOR) signaling pathway is considered as a central signaling hub coordinating eukaryotic cell growth and metabolism with environmental inputs. However, how and to what extent the TOR signaling pathway and rapamycin are involved in cellulase production remain elusive. RESULT: At the early fermentation stage, high-dose rapamycin (100 µM) caused a temporary inhibition effect on cellulase production, cell growth and sporulation of Trichoderma reesei RUT-C30 independently of the carbon sources, and specifically caused a tentative morphology defect in RUT-C30 grown on cellulose. On the contrary, the lipid content of T. reesei RUT-C30 was not affected by rapamycin. Accordingly, the transcriptional levels of genes involved in the cellulase production were downregulated notably with the addition of rapamycin. Although the mRNA levels of the putative rapamycin receptor trFKBP12 was upregulated significantly by rapamycin, gene trTOR (the downstream effector of the rapamycin-FKBP12 complex) and genes associated with the TOR signaling pathways were not changed markedly. With the deletion of gene trFKBP12, there is no impact of rapamycin on cellulase production, indicating that trFKBP12 mediates the observed temporary inhibition effect of rapamycin. CONCLUSION: Our study shows for the first time that only high-concentration rapamycin induced a transient impact on T. reesei RUT-C30 at its early cultivation stage, demonstrating T. reesei RUT-C30 is highly resistant to rapamycin, probably due to that trTOR and its related signaling pathways were not that sensitive to rapamycin. This temporary influence of rapamycin was facilitated by gene trFKBP12. These findings add to our knowledge on the roles of rapamycin and the TOR signaling pathways play in T. reesei.

Glutamine involvement in nitrogen regulation of cellulase production in fungi.

BACKGROUND: Cellulase synthesized by fungi can environment-friendly and sustainably degrades cellulose to fermentable sugars for producing cellulosic biofuels, biobased medicine and fine chemicals. Great efforts have been made to study the regulation mechanism of cellulase biosynthesis in fungi with the focus on the carbon sources, while little attention has been paid to the impact and regulation mechanism of nitrogen sources on cellulase production. RESULTS: Glutamine displayed the strongest inhibition effect on cellulase biosynthesis in Trichoderma reesei, followed by yeast extract, urea, tryptone, ammonium sulfate and L-glutamate. Cellulase production, cell growth and sporulation in T. reesei RUT-C30 grown on cellulose were all inhibited with the addition of glutamine (a preferred nitrogen source) with no change for mycelium morphology. This inhibition effect was attributed to both L-glutamine itself and the nitrogen excess induced by its presence. In agreement with the reduced cellulase production, the mRNA levels of 44 genes related to the cellulase production were decreased severely in the presence of glutamine. The transcriptional levels of genes involved in other nitrogen transport, ribosomal biogenesis and glutamine biosynthesis were decreased notably by glutamine, while the expression of genes relevant to glutamate biosynthesis, amino acid catabolism, and glutamine catabolism were increased noticeably. Moreover, the transcriptional level of cellulose signaling related proteins ooc1 and ooc2, and the cellular receptor of rapamycin trFKBP12 was increased remarkably, whose deletion exacerbated the cellulase depression influence of glutamine. CONCLUSION: Glutamine may well be the metabolite effector in nitrogen repression of cellulase synthesis, like the role of glucose plays in carbon catabolite repression. Glutamine under excess nitrogen condition repressed cellulase biosynthesis significantly as well as cell growth and sporulation in T. reesei RUT-C30. More importantly, the presence of glutamine notably impacted the transport and metabolism of nitrogen. Genes ooc1, ooc2, and trFKBP12 are associated with the cellulase repression impact of glutamine. These findings advance our understanding of nitrogen regulation of cellulase production in filamentous fungi, which would aid in the rational design of strains and fermentation strategies for cellulase production in industry.

Dissecting Cellular Function and Distribution of ß-Glucosidases in Trichoderma reesei.

Trichoderma reesei has 11 putative ß-glucosidases in its genome, playing key parts in the induction and production of cellulase. Nevertheless, the reason why the T. reesei genome encodes so many ß-glucosidases and the distinct role each ß-glucosidase plays in cellulase production remain unknown. In the present study, the cellular function and distribution of 10 known ß-glucosidases (CEL3B, CEL3E, CEL3F, CEL3H, CEL3J, CEL1A, CEL3C, CEL1B, CEL3G, and CEL3D) were explored in T. reesei, leaving out BGL1 (CEL3A), which has been well investigated. We found that the overexpression of cel3b or cel3g significantly enhanced extracellular ß-glucosidase production, whereas the overexpression of cel1b severely inhibited cellulase production by cellulose, resulting in nearly no growth of T. reesei Four types of cellular distribution patterns were observed for ß-glucosidases in T. reesei: (i) CEL3B, CEL3E, CEL3F, and CEL3G forming clearly separated protein secretion vesicles in the cytoplasm; (ii) CEL3H and CEL3J diffusing the whole endomembrane as well as the cell membrane with protein aggregation, like a reticular network; (iii) CEL1A and CEL3D in vacuoles; (iv) and CEL3C in the nucleus. ß-glucosidases CEL1A, CEL3B, CEL3E, CEL3F, CEL3G, CEL3H, and CEL3J were identified as extracellular, CEL3C and CEL3D as intracellular, and CEL1B as unknown. The extracellular ß-glucosidases CEL3B, CEL3E, CEL3F, CEL3H, and CEL3G were secreted through a tip-directed conventional secretion pathway, and CEL1A, via a vacuole-mediated pathway that was achieved without any signal peptide, while CEL3J was secreted via an unconventional protein pathway bypassing the endoplasmic reticulum (ER) and Golgi.IMPORTANCE Although ß-glucosidases play an important role in fungal cellulase induction and production, our current understanding does not provide a global perspective on ß-glucosidase function. This work comprehensively studies all the ß-glucosidases regarding their effect on cellulase production and their cellular distribution and secretion. Overexpression of cel3b or cel3g significantly enhanced ß-glucosidase production, whereas overexpression of cel1b severely inhibited cellulase production on cellulose. In addition, overexpression of cel3b, cel3e, cel3f, cel3h, cel3j, cel3c, or cel3g delayed endoglucanase (EG) production. We first identified four cellular distribution patterns of ß-glucosidases in Trichoderma reesei Specially, CEL3C was located in the nucleus. CEL3J was secreted through the nonclassical protein secretion pathway bypassing endoplasmic reticulum (ER) and Golgi. CEL1A was secreted via a vacuole-mediated conventional secretion route without a signal peptide. These findings advance our understanding of ß-glucosidase properties and secretory pathways in filamentous fungi, holding key clues for future study.

A polyp-on-chip for coral long-term culture.

Coral polyps are basic clonal biological units of reef corals. However, in vitro experimental model for long-term physiological and ecological studies has not been well developed due to the difficulty of effectively acquiring and culturing single polyps. This study developed an experimental platform based on microfluidics for culturing single coral polyps and tracing its growth state over time in the long run. The corresponding computational modeling was conducted to predict the metabolic processes under the static and dynamic conditions by coupling the mass transfer and reaction with Navier-Stokes equations. Design and fabrication of the microfluidic chip was the key to provide a constant laminar flow environment that enabled the controlled high oxygen and bicarbonate transfer for the cultivation of the single coral polyps. The single coral polyps were induced to bail out of the coral reef upon the chemical stress and cultured for more than fifteen days in the microfluidic chip. It was found that the single coral polyps in the microfluidic chip can maintain their normal metabolic process over the cultivation period, suggesting that our microfluidic platform can serve as a suitable tool to study the coral polyps by providing a controllable and suitable biological microenvironment.

Tracking localization and secretion of cellulase spatiotemporally and directly in living Trichoderma reesei.

BACKGROUND: Filamentous fungi secret hydrolytic enzymes like cellulase and hemicellulase outside the cells, serving as important scavengers of plant biomass in nature and workhorses in the enzyme industry. Unlike the extensive study on the mechanism of cellulase production in fungi, research on spatiotemporal distribution and secretion of cellulase in fungi is lacking, retarding the deeper understanding of the molecular mechanism behind the fungal cellulase production. RESULT: Recombinant Trichoderma reesei strains RBGL, RCBH, and RCMC were successfully constructed from T. reesei RUT-C30, expressing red fluorescent protein DsRed-tagged versions of ß-glucosidase (BGL), cellobiohydrolase (CBH), and endoglucanase (CMC), respectively. With the assistance of these strains, we found that all three cellulase components BGL, CBH, and CMC diffused throughout the whole fungal mycelium with major accumulation at the hyphal apexes. These enzymes located in ER, Golgi, vacuoles and cell membrane/wall, but not septum, and secreted abundantly into the culture medium. Moreover, the major secretion of CBH and CMC started more early than that of BGL. Brefeldin A (BFA) completely blocked cellulase expression and secretion in T. reesei. CONCLUSION: Based on recombinant T. reesei RBGL, RCBH, and RCMC expressing DsRed-fused versions of BGL, CBH, and CMC, respectively, the distribution and secretion of cellulase production in T. reesei were first visualized directly in a dynamic way, preliminarily mapping the location and secretion of T. reesei cellulase and providing evidence for revealing the secretion pathways of cellulase in T. reesei. The obtained results suggest that cellulase excretion majorly occurs via the conventional ER-Golgi secretory pathway, and might be assisted through unconventional protein secretion pathways.