Heterogenetic mechanism in high-temperature Daqu fermentation by traditional craft and mechanical craft: From microbial assembly patterns to metabolism phenotypes.

The mechanical process has a widely usage in large-scale high-temperature Daqu (HTD) enterprises, however, the quality of the mechanical HTD is gapped with the HTD by traditional process. Currently, the understanding of the mechanism behind this phenomenon is still over-constrained. To this end, the discrepancies in fermentation parameters, enzymatic characteristics, microbial assembly and succession patterns, metabolic phenotypes were compared between traditional HTD and mechanical HTD in this paper. The results showed that mechanical process altered the temperature ramping procedure, resulting in a delayed appearance of the peak temperature. This alteration shifted the assembly pattern of the initial bacterial community from determinism to stochasticity, while having no impact on the stochastic assembly pattern of the fungal community. Concurrently, mechanical pressing impeded the accumulation of arginase, tetramethylpyrazine, trimethylpyrazine, 2-methoxy-4-vinylphenol, and butyric acid, as the target dissimilarities in metabolism between traditional HTD and mechanical HTD. Pearson correlation analysis combined with the functional prediction further demonstrated that Bacillus, Virgibacillus, Oceanobacillus, Kroppenstedtia, Lactobacillus, and Monascus were mainly contributors to metabolic variances. The Redundancy analysis (RDA) of fermented environmental factors on functional ASVs indicated that high temperature, high acid and low moisture were key positive drivers on the microbial metabolism for the characteristic flavor in HTD. Based on these results, heterogeneous mechanisms between traditional HTD and mechanical HTD were explored, and controllable metabolism targets were as possible strategies to improve the quality of mechanical HTD.

Unraveling the chemosensory characteristics dependence of sauce-flavor baijiu on regionality using descriptive sensory analysis and quantitative targeted flavoromics.

Descriptive sensory analysis, headspace solid-phase microextraction-gas chromatography-mass spectrometry, gas chromatography-flame ionization detector and multivariate statistical analysis were used to elucidate the regional dependence of sauce-flavor baijiu (SFB). Although SFB samples from different regions couldn't be clearly classified by sensory profiles, they could be clearly divided into 5 groups in principal component analysis plot based on quantitative targeted flavoromics analysis. And then, the relationship between sensory attributes and volatile compounds were investigated by network analysis. Twenty regional aroma markers were identified by multivariate statistical analysis to distinguish SFB samples from different regions. Furthermore, the influence of manufacturing operation on SFB in Guizhou region was further analyzed. Thirty-eight potential compounds were significant different in Guizhou SFB samples with different manufacturing operations. This study not only provides a better understanding of regional dependence on SFB flavor, but also further clarifies the inheritance importance of manufacturing operation in traditional SFB production.

Mendelian randomization analysis reveals causality of inflammatory bowel disease on risks of Henoch-Schönlein purpura and immune thrombocytopenia.

BACKGROUND: Emerging clinical evidence has been discovered associating Inflammatory bowel disease (IBD) with Henoch-Schönlein purpura (HSP) and immune thrombocytopenia (ITP). However, it is unclear whether a cause-effect relationship exists between them. We aimed to examine the casual effect of IBD on the risk of HSP and ITP. METHODS: Based on summary statistics from International IBD Genetics (IIBDG) Consortium and FinnGen study, a two-sample Mendelian randomization study was carried out to determine whether IBD including ulcerative colitis (UC) and Crohn's disease (CD) is causally related to HSP, ITP or secondary thrombocytopenia. To support the results, a variety of sensitivity analyses were performed. RESULTS: Significant causal relationships between IBD and HSP (odds ratios = 1.20, 95% confidence interval: 1.07-1.36, adjusted P = 0.006) and ITP (odds ratios =1.22, 95% confidence interval: 1.08-1.38, adjusted P = 0.006) were found. Both genetically predicted UC and CD were positively related with ITP, while CD alone may be responsible for the higher risk of HSP. Besides, no significant association was observed between IBD and secondary thrombocytopenia. CONCLUSIONS: The results of this Mendelian randomization study supported the causal association of IBD with HSP and ITP. Taken together, our findings may present implications for management of IBD.

Using in situ untargeted flavoromics analysis to unravel the empty cup aroma of Jiangxiang-type Baijiu: A novel strategy for geographical origin traceability.

"Empty cup aroma" is an important characteristic and quality evaluation standard of Jiangxiang-type Baijiu (JXB). In this study, an in situ detection method for the empty cup aroma of JXB was established, and the authenticity and origin information of JXB were identified with an untargeted flavoromics strategy. The complex composition of JXB leads to slow ethanol volatilization, which is a potential method for identifying artificial JXB. The results of the sensory analysis showed that acidic, sauce, burnt and qu in the empty cup of JXB were the strongest at the 45 min stage. A total of 155 compounds were detected in the empty cups of 15 JXB from different regions during 45 min of standing, and 34 compounds were identified as key aroma compounds in the empty cups of JXB. Eleven potential markers were screened (VIP > 1), which can be used to distinguish JXB produced in Guizhou/Sichuan and other regions.

Improving Methanol Utilization by Reducing Alcohol Oxidase Activity and Adding Co-Substrate of Sodium Citrate in Pichia pastoris.

Methanol, which produced in large quantities from low-quality coal and the hydrogenation of CO2, is a potentially renewable one-carbon (C1) feedstock for biomanufacturing. The methylotrophic yeast Pichia pastoris is an ideal host for methanol biotransformation given its natural capacity as a methanol assimilation system. However, the utilization efficiency of methanol for biochemical production is limited by the toxicity of formaldehyde. Therefore, reducing the toxicity of formaldehyde to cells remains a challenge to the engineering design of a methanol metabolism. Based on genome-scale metabolic models (GSMM) calculations, we speculated that reducing alcohol oxidase (AOX) activity would re-construct the carbon metabolic flow and promote balance between the assimilation and dissimilation of formaldehyde metabolism processes, thereby increasing the biomass formation of P. pastoris. According to experimental verification, we proved that the accumulation of intracellular formaldehyde can be decreased by reducing AOX activity. The reduced formaldehyde formation upregulated methanol dissimilation and assimilation and the central carbon metabolism, which provided more energy for the cells to grow, ultimately leading to an increased conversion of methanol to biomass, as evidenced by phenotypic and transcriptome analysis. Significantly, the methanol conversion rate of AOX-attenuated strain PC110-AOX1-464 reached 0.364 g DCW/g, representing a 14% increase compared to the control strain PC110. In addition, we also proved that adding a co-substrate of sodium citrate could further improve the conversion of methanol to biomass in the AOX-attenuated strain. It was found that the methanol conversion rate of the PC110-AOX1-464 strain with the addition of 6 g/L sodium citrate reached 0.442 g DCW/g, representing 20% and 39% increases compared to AOX-attenuated strain PC110-AOX1-464 and control strain PC110 without sodium citrate addition, respectively. The study described here provides insight into the molecular mechanism of efficient methanol utilization by regulating AOX. Reducing AOX activity and adding sodium citrate as a co-substrate are potential engineering strategies to regulate the production of chemicals from methanol in P. pastoris.

Genome-wide analysis reveals Hsf1 maintains high transcript abundance of target genes controlled by strong constitutive promoter in Saccharomyces cerevisiae.

BACKGROUND: In synthetic biology, the strength of promoter elements is the basis for precise regulation of target gene transcription levels, which in turn increases the yield of the target product. However, the results of many researches proved that excessive transcription levels of target genes actually reduced the yield of the target product. This phenomenon has been found in studies using different microorganisms as chassis cells, thus, it becomes a bottleneck problem to improve the yield of the target product. RESULTS: In this study, promoters PGK1p and TDH3p with different strengths were used to regulate the transcription level of alcohol acetyl transferase encoding gene ATF1. The results demonstrated that the strong promoter TDH3p decreased the production of ethyl acetate. The results of Real-time PCR proved that the transcription level of ATF1 decreased rapidly under the control of TDH3p, and the unfolded protein reaction was activated, which may be the reason for the abnormal production caused by the strong promoter. RNA-sequencing analysis showed that the overexpression of differential gene HSP30 increased the transcriptional abundance of ATF1 gene and production of ethyl acetate. Interestingly, deletion of the heat shock protein family (e.g., Hsp26, Hsp78, Hsp82) decreased the production of ethyl acetate, suggesting that the Hsp family was also involved in the regulation of ATF1 gene transcription. Furthermore, the results proved that the Hsf1, an upstream transcription factor of Hsps, had a positive effect on alleviating the unfolded protein response and that overexpression of Hsf1 reprogramed the pattern of ATF1 gene transcript levels. The combined overexpression of Hsf1 and Hsps further increased the production of ethyl acetate. In addition, kinase Rim15 may be involved in this regulatory pathway. Finally, the regulation effect of Hsf1 on recombinant strains constructed by other promoters was verified, which confirmed the universality of the strategy. CONCLUSIONS: Our results elucidated the mechanism by which Rim15-Hsf1-Hsps pathway reconstructed the repression of high transcription level stress and increased the production of target products, thereby providing new insights and application strategies for the construction of recombinant strains in synthetic biology.

Analysis of the Effect of Mixed Fermentation on the Quality of Distilled Jujube Liquor by Gas Chromatography-Ion Mobility Spectrometry and Flavor Sensory Description.

Distilled jujube liquor is an alcoholic beverage made from jujube, which has a unique flavor and a sweet taste. The purpose of this study was to explore the effect of mixed fermentation on the quality of distilled jujube liquor by comparing the performance of mixed fermentation between S. cerevisiae, Pichia pastoris and Lactobacillus. The results showed that there were significant differences in the quality of the jujube liquor between the combined strains. Moreover, Lactobacillus increased and P. pastoris reduced the total acid content. The results from an E-nose showed that the contents of methyl, alcohol, aldehyde, and ketone substances in the test bottle decreased significantly after decanting, while the contents of inorganic sulfide and organic sulfide increased. Fifty flavor compounds were detected, including nineteen esters, twelve alcohols, seven ketones, six aldehydes, three alkenes, one furan, one pyridine, and one acid. There were no significant differences in the type or content of flavor compounds. However, PLS-DA showed differences among the samples. Eighteen volatile organic compounds with variable importance in projection values > 1 were obtained. There were sensory differences among the four samples. Compared with the sample fermented with only S. cerevisiae, the samples co-fermented with Lactobacillus or with P. pastoris had an obvious bitter taste and mellow taste, respectively. The sample fermented by all three strains had a prominent fruity flavor. Except for the sample fermented with only S. cerevisiae, the jujube flavor was weakened to varying degrees in all samples. Co-fermentation could be a valuable method to improve the flavor quality of distilled jujube liquor. This study revealed the effects of different mixed fermentation modes on the sensory flavor of distilled jujube liquor and provided a theoretical basis for the establishment of special mixed fermentation agents for distilled jujube liquor in the future.

The eisosomes contribute to acid tolerance of yeast by maintaining cell membrane integrity.

Microbes have evolved multiple mechanisms to resist environmental stresses, which are regulated in complex and delicate ways. Though the role of cell membranes in acid resistance from the perspective of physicochemical properties and membrane proteins has been deeply studied, the function of eisosomes is still in its infancy. In this study, we firstly reported the dynamic changes of eisosomes under acid stress and the decreased acid tolerance of yeasts caused by eisosome disruption. Physiological indicators and non-targeted lipid profiling revealed that eisosome disruption caused changes in multiple lipids and imbalances in lipid homeostasis, which are responsible for membrane integrity damage. Thus the increased infiltration of carboxylic acids and the raised ROS levels were detected in strains with disrupted eisosome assembly, resulting in decreased cellular tolerance. The results here provide novel insights into the acid-resistant mechanism of yeasts from the perspective of the cell membrane subdomain, which has practical impacts on green biological manufacturing and food preservation.

MtTRC-1, a Novel Transcription Factor, Regulates Cellulase Production via Directly Modulating the Genes Expression of the Mthac-1 and Mtcbh-1 in Myceliophthora thermophila.

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. IMPORTANCE In the present study, we characterized a novel regulator MtTRC-1 in M. thermophila, which regulated cellulase production through direct transcriptional regulation of the Mthac-1 and Mtcbh-1 genes. Our data demonstrated that MtHAC-1 is a key factor for the cellulase secretion capacity of M. thermophila. Our data indicate that this thermophilic fungus regulates cellulase production through a multilevels network, in which the protein secretory pathway is modulated by MtHAC-1-dependent UPR pathway and the cellulase gene expression is directly regulated in parallel by transcription factors. The conservation of Mttrc1 in filamentous fungi suggests this mechanism may be exploited to engineer filamentous fungal cell factories capable of producing proteins on an industrial scale.

RCO-3 and COL-26 form an external-to-internal module that regulates the dual-affinity glucose transport system in Neurospora crassa.

BACKGROUND: Low- and high-affinity glucose transport system is a conserved strategy of microorganism to cope with environmental glucose fluctuation for their growth and competitiveness. In Neurospora crassa, the dual-affinity glucose transport system consists of a low-affinity glucose transporter GLT-1 and two high-affinity glucose transporters HGT-1/HGT-2, which play diverse roles in glucose transport, carbon metabolism, and cellulase expression regulation. However, the regulation of this dual-transporter system in response to environmental glucose fluctuation is not yet clear. RESULTS: In this study, we report that a regulation module consisting of a downstream transcription factor COL-26 and an upstream non-transporting glucose sensor RCO-3 regulates the dual-affinity glucose transport system in N. crassa. COL-26 directly binds to the promoter regions of glt-1, hgt-1, and hgt-2, whereas RCO-3 is an upstream factor of the module whose deletion mutant resembles the Δcol-26 mutant phenotypically. Transcriptional profiling analysis revealed that Δcol-26 and Δrco-3 mutants had similar transcriptional profiles, and both mutants had impaired response to a glucose gradient. We also showed that the AMP-activated protein kinase (AMPK) complex is involved in regulation of the glucose transporters. AMPK is required for repression of glt-1 expression in starvation conditions by inhibiting the activity of RCO-3. CONCLUSIONS: RCO-3 and COL-26 form an external-to-internal module that regulates the glucose dual-affinity transport system. Transcription factor COL-26 was identified as the key regulator. AMPK was also involved in the regulation of the dual-transporter system. Our findings provide novel insight into the molecular basis of glucose uptake and signaling in filamentous fungi, which may aid in the rational design of fungal strains for industrial purposes.

Increasing Yield of 2,3,5,6-Tetramethylpyrazine in Baijiu Through Saccharomyces cerevisiae Metabolic Engineering.

Baijiu is a traditional distilled beverage in China with a rich variety of aroma substances. 2,3,5,6-tetramethylpyrazine (TTMP) is an important component in Baijiu and has the function of promoting cardiovascular and cerebrovascular health. During the brewing of Baijiu, the microorganisms in jiuqu produce acetoin and then synthesize TTMP, but the yield of TTMP is very low. In this work, 2,3-butanediol dehydrogenase (BDH) coding gene BDH1 and another BDH2 gene were deleted or overexpressed to evaluate the effect on the content of acetoin and TTMP in Saccharomyces cerevisiae. The results showed that the acetoin synthesis of strain α5-D1B2 was significantly enhanced by disrupting BDH1 and overexpressing BDH2, leading to a 2.6-fold increase of TTMP production up to 10.55 mg/L. To further improve the production level of TTMP, the α-acetolactate synthase (ALS) of the pyruvate decomposition pathway was overexpressed to enhance the synthesis of diacetyl. However, replacing the promoter of the ILV2 gene with a strong promoter (PGK1p) to increase the expression level of the ILV2 gene did not result in further increased diacetyl, acetoin and TTMP production. Based on these evidences, we constructed the diploid strains AY-SB1 (ΔBDH1:loxP/ΔBDH1:loxP) and AY-SD1B2 (ΔBDH1:loxP-PGK1p-BDH2-PGK1t/ΔBDH1:loxP-PGK1p-BDH2-PGK1t) to ensure the fermentation performance of the strain is more stable in Baijiu brewing. The concentration of TTMP in AY-SB1 and AY-SD1B2 was 7.58 and 9.47 mg/L, respectively, which represented a 2.3- and 2.87-fold increase compared to the parental strain. This work provides an example for increasing TTMP production in S. cerevisiae by genetic engineering, and highlight a novel method to improve the quality and beneficial health attributes of Baijiu.

Engineering Saccharomyces cerevisiae for production of the valuable monoterpene d-limonene during Chinese Baijiu fermentation.

d-Limonene, a cyclic monoterpene, possesses citrus-like olfactory property and multi-physiological functions. In this study, the d-limonene synthase (tLS) from Citrus limon was codon-optimized and heterologously expressed in Saccharomyces cerevisiae. The metabolic flux of canonical pathway based on overexpressing endogenous geranyl diphosphate synthase gene (ERG20) and its variant ERG20F96W-N127W was strengthened for improvement d-limonene production in Chinese Baijiu. To further elevate production, we established an orthogonal pathway by introducing neryl diphosphate synthase 1 (tNDPS1) from Solanum lycopersicum. The results showed that expressing ERG20 and ERG20F96W-N127W could enhance d-limonene synthesis, while expressing heterologous NPP synthase gene significantly increase d-limonene formation. Furthermore, we constructed a tLS-tNDPS1 fusion protein, and the best strain yielded 9.8 mg/L d-limonene after optimizing the amino acid linker and fusion order, a 40% improvement over the free enzymes during Chinese Baijiu fermentation. Finally, under the optimized fermentation conditions, a maximum d-limonene content of 23.7 mg/L in strain AY12α-L9 was achieved, which was the highest reported production in Chinese Baijiu. In addition, we also investigated that the effect of d-limonene concentration on yeast growth and fermentation. This study provided a meaningful insight into the platform for other valuable monoterpenes biosynthesis in Chinese Baijiu fermentation.

Direct production of commodity chemicals from lignocellulose using Myceliophthora thermophila.

The production of fuels and chemicals from renewable plant biomass has been proposed as a feasible strategy for global sustainable development. However, the economic efficiency of biorefineries is low. Here, through metabolic engineering, Myceliophthora thermophila, a cellulolytic thermophilic fungus, was constructed into a platform that can efficiently convert lignocellulose into important bulk chemicals-four carbon 1, 4-diacids (malic and succinic acid), building blocks for biopolymers-without the need for extra hydrolytic enzymes. Titers of >200 g/L from crystalline cellulose and 110 g/L from plant biomass (corncob) were achieved during fed-batch fermentation. Our study represents a milestone in consolidated bioprocessing technology and offers a new and promising system for the cost-effective production of chemicals and fuels from biomass.

Broad Substrate-Specific Phosphorylation Events Are Associated With the Initial Stage of Plant Cell Wall Recognition in Neurospora crassa.

Fungal plant cell wall degradation processes are governed by complex regulatory mechanisms, allowing the organisms to adapt their metabolic program with high specificity to the available substrates. While the uptake of representative plant cell wall mono- and disaccharides is known to induce specific transcriptional and translational responses, the processes related to early signal reception and transduction remain largely unknown. A fast and reversible way of signal transmission are post-translational protein modifications, such as phosphorylations, which could initiate rapid adaptations of the fungal metabolism to a new condition. To elucidate how changes in the initial substrate recognition phase of Neurospora crassa affect the global phosphorylation pattern, phospho-proteomics was performed after a short (2 min) induction period with several plant cell wall-related mono- and disaccharides. The MS/MS-based peptide analysis revealed large-scale substrate-specific protein phosphorylation and de-phosphorylations. Using the proteins identified by MS/MS, a protein-protein-interaction (PPI) network was constructed. The variance in phosphorylation of a large number of kinases, phosphatases and transcription factors indicate the participation of many known signaling pathways, including circadian responses, two-component regulatory systems, MAP kinases as well as the cAMP-dependent and heterotrimeric G-protein pathways. Adenylate cyclase, a key component of the cAMP pathway, was identified as a potential hub for carbon source-specific differential protein interactions. In addition, four phosphorylated F-Box proteins were identified, two of which, Fbx-19 and Fbx-22, were found to be involved in carbon catabolite repression responses. Overall, these results provide unprecedented and detailed insights into a so far less well known stage of the fungal response to environmental cues and allow to better elucidate the molecular mechanisms of sensory perception and signal transduction during plant cell wall degradation.

STK-12 acts as a transcriptional brake to control the expression of cellulase-encoding genes in Neurospora crassa.

Cellulolytic fungi have evolved a complex regulatory network to maintain the precise balance of nutrients required for growth and hydrolytic enzyme production. When fungi are exposed to cellulose, the transcript levels of cellulase genes rapidly increase and then decline. However, the mechanisms underlying this bell-shaped expression pattern are unclear. We systematically screened a protein kinase deletion set in the filamentous fungus Neurospora crassa to search for mutants exhibiting aberrant expression patterns of cellulase genes. We observed that the loss of stk-12 (NCU07378) caused a dramatic increase in cellulase production and an extended period of high transcript abundance of major cellulase genes. These results suggested that stk-12 plays a critical role as a brake to turn down the transcription of cellulase genes to repress the overexpression of hydrolytic enzymes and prevent energy wastage. Transcriptional profiling analyses revealed that cellulase gene expression levels were maintained at high levels for 56 h in the Δstk-12 mutant, compared to only 8 h in the wild-type (WT) strain. After growth on cellulose for 3 days, the transcript levels of cellulase genes in the Δstk-12 mutant were 3.3-fold over WT, and clr-2 (encoding a transcriptional activator) was up-regulated in Δstk-12 while res-1 and rca-1 (encoding two cellulase repressors) were down-regulated. Consequently, total cellulase production in the Δstk-12 mutant was 7-fold higher than in the WT. These results strongly suggest that stk-12 deletion results in dysregulation of the cellulase expression machinery. Further analyses showed that STK-12 directly targets IGO-1 to regulate cellulase production. The TORC1 pathway promoted cellulase production, at least partly, by inhibiting STK-12 function, and STK-12 and CRE-1 functioned in parallel pathways to repress cellulase gene expression. Our results clarify how cellulase genes are repressed at the transcriptional level during cellulose induction, and highlight a new strategy to improve industrial fungal strains.

Auriculatone Sulfate Effectively Protects Mice Against Acetaminophen-Induced Liver Injury.

Acetaminophen (APAP) overdose is very common worldwide and has been widely recognized as the leading cause of drug-induced liver injury in the Western world. In our previous investigation, auriculatone, a natural product firstly obtained from Aster auriculatus, has demonstrated a potent protective effect against APAP-induced hepatotoxicity in HL-7702 cells. However, the poor water solubility and low bioavailability restrict its application. Auriculatone sulfate (AS) is a sulfated derivative of auriculatone with highly improved water-solubility. Hepatoprotective effects against APAP-induced liver injury (AILI) showed that intragastric pretreatment with AS at 50 mg/kg almost completely prevented mice against APAP-induced increases of serum alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase and ATPase. Histological results showed that AS could protect the liver tissue damage. In addition, AS pretreatment not only significantly retained hepatic malondialdehyde and the activities of glutathione, superoxide dismutase, and glutathione peroxidase at normal levels, but also markedly suppressed the increase of pro-inflammatory cytokines TNF-α, IL-1ß, and IL-6 levels in mouse liver caused by overdose APAP. Immunohistochemical analysis showed that AS obviously attenuated the expression of CD45 and HNE in liver tissue. Further mechanisms of action investigation showed that inhibition of cytochrome P450 3A11 (CYP 3A11) and CYP2E1 enzymatic activities (but not that of CYP1A2) was responsible for APAP bioactivation. In conclusion, AS showed a hepatoprotective effect against AILI through alleviating oxidative stress and inflammation and inhibiting CYP-mediated APAP bioactivation. It may be an effective hepatoprotective agent for AILI and other forms of human liver disease.

Crosstalk of Cellulose and Mannan Perception Pathways Leads to Inhibition of Cellulase Production in Several Filamentous Fungi.

It is essential for microbes to acquire information about their environment. Fungi use soluble degradation products of plant cell wall components to understand the substrate composition they grow on. Individual perception pathways have been well described. However, the interconnections between pathways remain poorly understood. In the present work, we provide evidence of crosstalk between the perception pathways for cellulose and the hemicellulose mannan being conserved in several filamentous fungi and leading to the inhibition of cellulase expression. We used the functional genomics tools available for Neurospora crassa to investigate this overlap at the molecular level. Crosstalk and competitive inhibition could be identified both during uptake by cellodextrin transporters and intracellularly. Importantly, the overlap is independent of CRE-1-mediated catabolite repression. These results provide novel insights into the regulatory networks of lignocellulolytic fungi and will contribute to the rational optimization of fungal enzyme production for efficient plant biomass depolymerization and utilization.IMPORTANCE In fungi, the production of enzymes for polysaccharide degradation is controlled by complex signaling networks. Previously, these networks were studied in response to simple sugars or single polysaccharides. Here, we tackled for the first time the molecular interplay between two seemingly unrelated perception pathways: those for cellulose and the hemicellulose (gluco)mannan. We identified a so far unknown competitive inhibition between the respective degradation products acting as signaling molecules. Competition was detected both at the level of the uptake and intracellularly, upstream of the main transcriptional regulator CLR-2. Our findings provide novel insights into the molecular communication between perception pathways. Also, they present possible targets for the improvement of industrial strains for higher cellulase production through the engineering of mannan insensitivity.

Disruption of gul-1 decreased the culture viscosity and improved protein secretion in the filamentous fungus Neurospora crassa.

BACKGROUND: The cellulolytic fungus Neurospora crassa is considered a potential host for enzyme and bioethanol production. However, large scale applications are hindered by its filamentous growth. Although previous investigations have shown that mycelial morphology in submerged culture can be controlled by altering physical factors, there is little knowledge available about the potential for morphology control by genetic modification. RESULTS: In this study, we screened morphological mutants in the filamentous fungus N. crassa. Of the 90 morphological mutants screened, 14 mutants exhibited considerably higher viscosity compared with that of the wild type strain, and only two mutants showed low-viscosity morphologies in submerged culture. We observed that disruption of gul-1 (NCU01197), which encodes an mRNA binding protein involved in cell wall remodeling, caused pellet formation as the fermentation progressed, and resulted in the most significant decrease in viscosity of culture broth. Moreover, over-expression of gul-1 caused dramatically increased viscosity, suggesting that the gul-1 had an important function in mycelial morphology during submerged cultivation. Transcriptional profiling showed that expression of genes encoding eight GPI-anchored cell wall proteins was lowered in Δgul-1 while expression of genes associated with two non-anchored cell wall proteins was elevated. Meanwhile, the expression levels of two hydrophobin genes were also significantly altered. These results suggested that GUL-1 affected the transcription of cell wall-related genes, thereby influencing cell wall structure and mycelial morphology. Additionally, the deletion of gul-1 caused increased protein secretion, probably due to a defect in cell wall integrity, suggesting this as an alternative strategy of strain improvement for enzyme production. To confirm practical applications, deletion of gul-1 in the hyper-cellulase producing strain (∆ncw-1∆Ncap3m) significantly reduced the viscosity of culture broth. CONCLUSIONS: Using the model filamentous fungus N. crassa, genes that affect mycelial morphology in submerged culture were explored through systematic screening of morphological mutants. Disrupting several candidate genes altered viscosities in submerged culture. This work provides an example for controlling fungal morphology in submerged fermentation by genetic engineering, and will be beneficial for industrial fungal strain improvement.

Development of a genome-editing CRISPR/Cas9 system in thermophilic fungal Myceliophthora species and its application to hyper-cellulase production strain engineering.

BACKGROUND: Over the past 3 years, the CRISPR/Cas9 system has revolutionized the field of genome engineering. However, its application has not yet been validated in thermophilic fungi. Myceliophthora thermophila, an important thermophilic biomass-degrading fungus, has attracted industrial interest for the production of efficient thermostable enzymes. Genetic manipulation of Myceliophthora is crucial for metabolic engineering and to unravel the mechanism of lignocellulose deconstruction. The lack of a powerful, versatile genome-editing tool has impeded the broader exploitation of M. thermophila in biotechnology. RESULTS: In this study, a CRISPR/Cas9 system for efficient multiplexed genome engineering was successfully developed in the thermophilic species M. thermophila and M. heterothallica. This CRISPR/Cas9 system could efficiently mutate the imported amdS gene in the genome via NHEJ-mediated events. As a proof of principle, the genes of the cellulase production pathway, including cre-1, res-1, gh1-1, and alp-1, were chosen as editing targets. Simultaneous multigene disruptions of up to four of these different loci were accomplished with neomycin selection marker integration via a single transformation using the CRISPR/Cas9 system. Using this genome-engineering tool, multiple strains exhibiting pronounced hyper-cellulase production were generated, in which the extracellular secreted protein and lignocellulase activities were significantly increased (up to 5- and 13-fold, respectively) compared with the parental strain. CONCLUSIONS: A genome-wide engineering system for thermophilic fungi was established based on CRISPR/Cas9. Successful expansion of this system without modification to M. heterothallica indicates it has wide adaptability and flexibility for use in other Myceliophthora species. This system could greatly accelerate strain engineering of thermophilic fungi for production of industrial enzymes, such as cellulases as shown in this study and possibly bio-based fuels and chemicals in the future.

Disruption of non-anchored cell wall protein NCW-1 promotes cellulase production by increasing cellobiose uptake in Neurospora crassa.

OBJECTIVES: To elucidate the mechanism of cellulase signal transduction in filamentous fungi including the components of the cellulase induction pathway. RESULTS: Neurospora crassa ncw-1 encodes a non-anchored cell wall protein. The absence of ncw-1 increased cellulase gene expression and this is not due to relieving carbon catabolite repression mediated by the cre-1 pathway. A mutant lacking genes encoding both three major ß-glucosidase enzymes and NCW-1 (Δ3ßGΔncw-1) was constructed. Transcriptome analysis of the quadruple mutant demonstrated enhanced expression of cellodextrin transporters after ncw-1 deletion, indicating that ncw-1 affects cellulase expression and production by inhibiting the uptake of the cellodextrin. CONCLUSIONS: NCW-1 is a novel component that plays a critical role in the cellulase induction signaling pathway.