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Background. 4-Methoxydalbergione (4MOD) is a flavonoid isolated from the heartwood of Dalbergia. Studies have demonstrated that 4MOD exerts anticancer activities on bladder cancer and astrocytoma. However, the anticancer activity of 4MOD in hepatocellular carcinoma (HCC) remains unknown. This study aims to examine its anticancer activities and mechanisms in human liver cancer cells. Methods. CCK-8, colony forming, wound healing, transwell migration, and AnnexinV/PI assays were used to assess the anticancer effects of 4MOD in HCC cells. RNA sequencing (RNA-Seq) was selected to explore the possible mechanisms underlying the anti-HCC activity of 4MOD. The mRNA expression levels of target genes were verified through quantitative real-time PCR (qRT-PCR). A lentiviral shRNA interference technique was used to silence GADD45G expression. GADD45G knockdown was employed to confirm the crucial role of GADD45G in the 4MOD-mediatedanti-HCC effects. Results. 4MOD inhibited HCC cells’ proliferation and migration and promoted tumor cell apoptosis. RNA-Seq and qRT-PCR analyses revealed that 4MOD treatment increased GADD45G expression. Silencing GADD45G reversed 4MOD-mediated inhibition of proliferation, migration, and promotion of apoptosis. Conclusions. Our findings show that 4MOD elicits anti-HCC effects by upregulating GADD45G expression and could be a valuable anticancer agent for liver cancer.

Efficient cellulolytic enzyme production is important for the development of lignocellulose-degrading enzyme mixtures. However, purification of cellulases from their native hosts is time- and labor-consuming. In this study, a constitutive expression system was developed in Penicillium oxalicum for the secreted production of proteins. Using a constitutive polyubiquitin gene promoter and cultivating with glucose as the sole carbon source, nine cellulolytic enzymes of different origins with relatively high purity were produced within 48 h. When supplemented to a commercial cellulase preparation, cellobiohydrolase I from P. funiculosum and cellobiohydrolase II from Talaromyces verruculosus showed remarkable enhancing effects on the hydrolysis of steam-exploded corn stover. Additionally, a synergistic effect was observed for these two cellobiohydrolases during the hydrolysis. Taken together, the constitutive expression system provides a convenient tool for the production of cellulolytic enzymes, which is expected to be useful in the development of highly efficient lignocellulose-degrading enzyme mixtures.

High efficiency enzymatic hydrolysis of lignocellulose to produce fermentable sugar is important for bioconversion of lignocellulosic biomass to liquid fuels and chemicals. In this study, a simple pretreatment with potassium permanganate is used to enhance the efficiency of organosolv pretreatment of corn stover. It is testified that, compared to the single organosolv pretreatment, the KMnO4 assisted organosolv pretreatment significantly increase yields of carbohydrates through decreasing degradation of hemicellulose, decrease formation of inhibitors and enhance enzymatic hydrolysis of corn stover pretreated at a low pretreatment temperature. Process conditions of the KMnO4 assisted organosolv pretreatment are optimized and the conversion of glucan and xylan in pretreated corn stover at 72 h of enzymatic hydrolysis reach to 89.45% and 85.30% respectively when corn stover is firstly treated with 1% (w/w) KMnO4 at 40°C for 60 min prior to ethanol‐water pretreatment with 50% ethanol concentration at 185°C for 90 min. The differences in morphological feature, fiber surface chemical compositions, crystallinity degree and thermostability of pretreated corn stover by ethanol‐water with and without KMnO4 are characterized by scanning electron microscope, X‐ray photoelectron spectroscopy, X‐ray diffraction and thermogravimetry to explore the underlying reasons of KMnO4 strengthening ethanol‐water pretreatment process.

Recent technological breakthroughs in single-particle cryo-electron microscopy (cryo-EM) enabled rapid atomic structure determination of biological macromolecules. A major bottleneck in the current single particle cryo-EM pipeline is to prepare good quality frozen cryo-EM grids, which often is a trial- and-error process. Among many issues, preferred particle orientation and sample damaging by air-water interface (AWI) are common problem in practice. Here we reported a method of applying metallo-supramolecular branched polymer (MSBP) in the cryo-sample preparation for high-resolution single-particle cryo-EM. Our data shows that applying MSBP keeps majority of particles away from air-water interface and mitigates preferred orientation, as demonstrated by analysis of both apoferritin and hemagglutinin (HA) trimer. Together, the use of MSBP is a simple method and could be applied to improve particle distribution for high-resolution structure determination using single-particle cryo-EM.

To construct convenient CRISPR/Cas9-mediated genome editing systems in industrial enzyme-producing fungi Penicillium oxalicum and Trichoderma reesei. Employing the 5S rRNA promoter from Aspergillus niger for guide RNA expression, the β-glucosidase gene bgl2 in P. oxalicum was deleted using a donor DNA carrying 40-bp homology arms or a donor containing no selectable marker gene. Using a markerless donor DNA as editing template, precise replacement of a small region was achieved in the creA gene. In T. reesei, the A. niger 5S rRNA promoter was less efficient than that in P. oxalicum when used for gene editing. Using a native 5S rRNA promoter, stop codons were introduced into the lae1 coding region using a markerless donor DNA with an editing efficiency of 36.67%. Efficient genome editing systems were developed in filamentous fungi P. oxalicum and T. reesei by using heterologous or native 5S rRNA promoters for guide RNA expression.

The degradation of lignocellulosic biomass by cellulolytic enzymes is involved in the global carbon cycle. The hydrolysis of lignocellulosic biomass into fermentable sugars is potential as an excellent industrial resource to produce a variety of chemical products. The production of cellulolytic enzymes is regulated mainly at the transcriptional level in filamentous fungi. Transcription factor ClrB and the putative histone methyltransferase LaeA, are both necessary for the expression of cellulolytic genes. However, the mechanism by which transcription factors and methyltransferase coordinately regulate cellulolytic genes is still unknown. Here, we reveal a transcriptional regulatory mechanism involving Penicillium oxalicum transcription factor ClrB (PoClrB), complex Tup1-Cyc8, and putative histone methyltransferase LaeA (PoLaeA). As the transcription factor, PoClrB binds the targeted promoters of cellulolytic genes, recruits PoTup1-Cyc8 complex via direct interaction with PoTup1. PoTup1 interacts with PoCyc8 to form the coactivator complex PoTup1-Cyc8. Then, PoTup1 recruits putative histone methyltransferase PoLaeA to modify the chromatin structure of the upstream region of cellulolytic genes, thereby facilitating the binding of transcription machinery to activating the corresponding cellulolytic gene expression. Our results contribute to a better understanding of complex transcriptional regulation mechanisms of cellulolytic genes and will be valuable for lignocellulosic biorefining.

Fungi sense environmental signals and coordinate growth, development, and metabolism accordingly. Calcium-calmodulin-calcineurin signaling is a conserved cascade pathway in fungi. One of the most important downstream targets of this pathway is the transcription factor Crz1/CrzA, which plays an essential role in various cellular processes. The putative collaborators of Penicillium oxalicum CrzA (PoCrzA) were found, through tandem affinity purification followed by mass spectrometric analysis (TAP-MS). A total of 50 protein-protein interaction collaborators of PoCrzA were observed. Among them, some collaborators, such as the catalytic subunit of calcineurin (Cna1, calcineurin A), the regulatory catalytic subunit of calcineurin (Cnb1, calcineurin B), and a 14-3-3 protein Bmh1, which were previously reported in yeast, were identified. Some putative collaborators, including two karyopherins (exportin Los1 and importin Srp1), two kinases (Fus3 and Slt2p), and a general transcriptional corepressor (Cyc8), were also found. The CrzA deletion mutant ΔPocrzA exhibited slow hyphal growth, impaired conidiogenesis, and reduced extracellular cellulase synthesis. Phenotype and transcriptome analysis showed that PoCrzA regulated fungal development in a Flbs-BrlA-dependent manner and participated in cellulase synthesis by modulating cellulolytic gene expression. On the basis of the results of TAP-MS, transcriptome, and phenotypic analysis in P. oxalicum, our study was the first to draft the calcineurin-CrzA pathway in cellulolytic fungi.

Enzymes that degrade lignocellulose to simple sugars are of great interest in research and for biotechnology because of their role in converting plant biomass to fuels and chemicals. The synthesis of cellulolytic enzymes in filamentous fungi is tightly regulated at the transcriptional level, with the transcriptional activator ClrB/CLR-2 playing a critical role in many species. In Penicillium oxalicum, clrB overexpression could not relieve the dependence of cellulase expression on cellulose as an inducer, suggesting that clrB is controlled post-transcriptionally. In this study, using a reporter gene system in yeast, we identified the C-terminal region of ClrB/CLR-2 as a transcriptional activation domain. Expression of clrBID, encoding a ClrB derivative in which the DNA-binding and transcriptional activation domains are fused together to remove the middle region, led to cellulase production in the absence of cellulose in P. oxalicum. Strikingly, the clrBID-expressing strain produced cellulase on carbon sources that normally repress cellulase expression, including glucose and glycerol. Results from deletion of the carbon catabolite repressor gene creA in the clrBID-expressing strain suggested that the effect of clrBID is independent of CreA's repressive function. A similar modification of clrB in Aspergillus niger resulted in the production of a mannanase in glucose medium. Taken together, these results indicate that ClrB suppression under noninducing conditions involves its middle region, suggesting a potential strategy to engineer fungal strains for improved cellulase production on commonly used carbon sources.

CpcA is a conserved transcriptional activator for the cross-pathway control of amino acid biosynthetic genes in filamentous fungi. Previous studies of this regulator mainly revealed its function under amino acid starvation condition, where amino acid biosynthetic inhibitors were added in the culture. In this study, the biological function of CpcA in Penicillium oxalicum was investigated under different cultivation conditions. Disruption of cpcA led to decreased cell growth either in the presence or absence of histidine biosynthetic inhibitor, and the phenotype could be rescued by the addition of exogenous amino acid sources. In addition, CpcA was required for the rapid production of cellulase when cells were cultured on cellulose. Transcript abundance measurement showed that a set of amino acid biosynthetic genes as well as two major cellulase genes were significantly down-regulated in cpcA deletion mutant relative to wild type. Taken together, the results revealed the biological role of CpcA in supporting normal growth and extracellular enzyme production of P. oxalicum under amino acid non-starvation condition.

PoCel12A, PoCel12B, and PoCel12C are genes that encode glycoside hydrolase family 12 (GH12) enzymes in Penicillium oxalicum. PoCel12A and PoCel12B are typical GH12 enzymes that belong to fungal subfamilies 12-1 and 12-2, respectively. PoCel12C contains a low-complexity region (LCR) domain, which is not found in PoCel12A or PoCel12B and independent of fungal subfamily 12-1 or 12-2. Recombinant enzymes (named rCel12A, rCel12B and rCel12C) demonstrate existing diversity in the substrate specificities. Although most members in GH family 12 are typical endoglucanases and preferentially hydrolyze β-1,4-glucan (e.g., carboxymethylcellulose), recombinant PoCel12A is a non-typical endo-(1-4)-β-glucanase; it preferentially hydrolyzes mix-linked β-glucan (barley β-glucan, β-1,3-1,4-glucan) and slightly hydrolyzes β-1,4-glucan (carboxymethylcellulose). Recombinant PoCel12B possesses a significantly high activity against xyloglucan. A specific activity of rCel12B toward xyloglucan (239 µmol/min/mg) is the second-highest value known. Recombinant PoCel12C shows low activity toward β-glucan, carboxymethylcellulose, or xyloglucan. All three enzymes can degrade phosphoric acid-swollen cellulose (PASC). However, the hydrolysis products toward PASC by enzymes are different: the main hydrolysis products are cellotriose, cellotetraose, and cellobiose for rCel12A, rCel12B, and rCel12C, correspondingly. A synergistic action toward PASC among rCel12A and rCel12B is observed, thereby suggesting a potential application for preparing enzyme cocktails used in lignocellulose hydrolysis.

There is a rising interest in bioethanol production from lignocellulose such as corn stover to decrease the need for fossil fuels, but most research mainly focuses on how to improve ethanol yield and pays less attention to the biorefinery of corn stover. To realize the utilization of different components of corn stover in this study, different pretreatment strategies were used to fractionate corn stover while enhancing enzymatic digestibility and cellulosic ethanol production. It was found that the pretreatment process combining dilute acid (DA) and alkaline sodium sulfite (ASS) could effectively fractionate the three main components of corn stover, i.e., cellulose, hemicellulose, and lignin, that xylose recovery reached 93.0%, and that removal rate of lignin was 85.0%. After the joint pretreatment of DA and ASS, the conversion of cellulose at 72 h of enzymatic hydrolysis reached 85.4%, and ethanol concentration reached 48.5 g/L through fed-batch semi-simultaneous saccharification and fermentation (S-SSF) process when the final concentration of substrate was 18% (w/v). Pretreatment with ammonium sulfite resulted in 83.8% of lignin removal, and the conversion of cellulose and ethanol concentration reached 86.6% and 50 g/L after enzymatic hydrolysis of 72 h and fed-batch S-SSF, respectively. The results provided a reference for effectively separating hemicellulose and lignin from corn stover and producing cellulosic ethanol for the biorefinery of corn stover.

Next basket recommendation aims to infer a set of items that a user will purchase at the next visit by considering a sequence of baskets he/she has purchased previously. This task has drawn increasing attention from both the academic and industrial communities. The existing solutions mainly focus on sequential modeling over their historical interactions. However, due to the diversity and randomness of users' behaviors, not all these baskets are relevant to help identify the user's next move. It is necessary to denoise the baskets and extract credibly relevant items to enhance recommendation performance. Unfortunately, this dimension is usually overlooked in the current literature. To this end, in this paper, we propose a Contrastive Learning Model~(named CLEA) to automatically extract items relevant to the target item for next basket recommendation. Specifically, empowered by Gumbel Softmax, we devise a denoising generator to adaptively identify whether each item in a historical basket is relevant to the target item or not. With this process, we can obtain a positive sub-basket and a negative sub-basket for each basket over each user. Then, we derive the representation of each sub-basket based on its constituent items through a GRU-based context encoder, which expresses either relevant preference or irrelevant noises regarding the target item. After that, a novel two-stage anchor-guided contrastive learning process is then designed to simultaneously guide this relevance learning without requiring any item-level relevance supervision. To the best of our knowledge, this is the first work of performing item-level denoising for a basket in an end-to-end fashion for next basket recommendation. Extensive experiments are conducted over four real-world datasets with diverse characteristics. The results demonstrate that our proposed CLEA achieves significantly better recommendation performance than the existing state-of-the-art alternatives. Moreover, further analysis also shows that CLEA can successfully discover the real relevant items towards the recommendation decision.

Hyphal morphology is considered to have a close relationship with the production level of secreted proteins by filamentous fungi. In this study, the gul1 gene, which encodes a putative mRNA-binding protein, was disrupted in cellulase-producing fungus Trichoderma reesei. The hyphae of Δgul1 strain produced more lateral branches than the parent strain. Under the condition for cellulase production, disruption of gul1 resulted in smaller mycelial clumps and significantly lower viscosity of fermentation broth. In addition, cellulase production was improved by 22% relative to the parent strain. Transcriptome analysis revealed that a set of genes encoding cell wall remodeling enzymes as well as hydrophobins were differentially expressed in the Δgul1 strain. The results suggest that the regulatory role of gul1 in cell morphogenesis is likely conserved in filamentous fungi. To our knowledge, this is the first report on the engineering of gul1 in an industrially important fungus.

Efficient cellulolytic enzyme production is important for the development of lignocellulose-degrading enzyme mixtures. However, purification of cellulases from their native hosts is time- and labor-consuming. In this study, a constitutive expression system was developed in

As the universal methyl donor for methylation reactions, S-adenosylmethionine (AdoMet) plays an indispensable role in most cellular metabolic processes. AdoMet is synthesized by AdoMet synthetase. We identified the only one AdoMet synthetase (PoSasA) in filamentous fungus Penicillium oxalicum. PoSasA was widely distributed in mycelium at different growth stages. The absence of PoSasA was lethal for P. oxalicum. The misregulation of the PoSasA encoding gene affected the synthesis of extracellular cellulolytic enzymes. The expression levels of cellobiohydrolase encoding gene cbh1/cel7A, β-1-4 endoglucanase eg1/cel7B, and xylanase encoding gene xyn10A were remarkably downregulated as a result of decreased PosasA gene expression. The production of extracellular cellulases and hemicellulases was also reduced. By contrast, the overexpression of PosasA improved the production of extracellular cellulases and hemicellulases. A total of 133 putative interacting proteins with PoSasA were identified using tandem affinity purification and mass spectrometry. The results of functional enrichment on these proteins showed that they were mainly related to ATP binding, magnesium ion binding, and ATP synthetase activity. Several methyltransferases were also observed among these proteins. These results were consistent with the intrinsic feature of AdoMet synthetase. This work reveals the indispensable role of PoSasA in various biological processes.

Machine learning algorithm is the core of artificial intelligence, is the fundamental way to make computer intelligent, its application in all fields of artificial intelligence. Aiming at the problems of the existing algorithms in the discrete manufacturing industry, this paper proposes a new 0-1 coding method to optimize the learning algorithm, and finally proposes a learning algorithm of “IG type learning only from the best”.

Spent ammonium sulphite liquor (SASL) is the main effluent from the ammonium sulphite pulping process, and contains amounts of lignocellulosic oligomers, monosaccharides and ammonium salts. The effect of continuous SASL-feeding on cellulase production by Penicillium oxalicum was studied. With a rate-varying feeding strategy, the maximal filter paper enzyme (FPase) activity reached 17.66U/mL at 144h, and the specific FPase activity increased from 1.74U/mg (without SASL) to 2.40U/mg. Accordingly, the glucan hydrolysis conversion of delignified corn cob residue by the enzymes from continuous SASL-feeding fermentation was significantly higher than that without SASL at equal protein loadings. Comparative proteomic analysis demonstrated that the proteins involved in lignocellulose degradation were specifically up-regulated in the crude cellulase with SASL-feeding. The obtained crude enzyme was efficient in the hydrolysis of pulping products, with a glucan conversion of 81.87% achieved after 72h saccharification of ammonium sulphite pulp.

Efficient deconstruction of lignocellulose is achieved by the synergistic action of various hydrolytic and oxidative enzymes. However, the aldonolactones generated by oxidative enzymes have inhibitory effects on some cellulolytic enzymes. In this work, D-glucono-1,5-lactone was shown to have a much stronger inhibitory effect than D-glucose and D-gluconate on β-glucosidase, a vital enzyme during cellulose degradation. AltA, a secreted enzyme from Penicillium oxalicum, was identified as an aldonolactonase which can catalyze the hydrolysis of D-glucono-1,5-lactone to D-gluconic acid. In the course of lignocellulose saccharification conducted by cellulases from P. oxalicum or Trichoderma reesei, supplementation of AltA was able to relieve the decrease of β-glucosidase activity obviously with a stimulation of glucose yield. This boosting effect disappeared when sodium azide and ethylenediaminetetraacetic acid (EDTA) were added to the saccharification system to inhibit the activities of oxidative enzymes. In summary, we describe the first heterologous expression of a fungal secreted aldonolactonase and its application as an efficient supplement of cellulolytic enzyme system for lignocellulose biodegradation.

Putative methyltranferase LaeA and LaeA-like proteins, which are conserved in many filamentous fungi, regulate the sporogenesis and biosynthesis of secondary metabolites. In this study, we reported the biological function of a LaeA-like methyltransferase, Penicillium oxalicum Mtr23B, which contains a methyltransf_23 domain and an S-adenosylmethionine binding domain, in controlling spore pigment formation and in the expression of secondary metabolic gene cluster and glycoside hydrolase genes. Additionally, we compared Mtr23B and LaeA, and determined their similarities and differences in terms of their roles in regulating the above biological processes. mtr23B had the highest transcriptional level among the 12 members of the methyltransf_23 family in P. oxalicum. The colony color of Δmtr23B (deletion of mtr23B) was lighter than that of ΔlaeA, although Δmtr23B produced ~ 19.2-fold more conidia than ΔlaeA. The transcriptional levels of abrA, abrB/yA, albA/wA, arpA, arpB, and aygA, which are involved in the dihydroxynaphtalene–melanin pathway, decreased in Δmtr23B. However, Mtr23B had a little effect on brush-like structures and conidium formation, and had a different function from LaeA. Mtr23B extensively regulated glycoside hydrolase gene expression. The absence of Mtr23B remarkably repressed prominent cellulase- and amylase-encoding genes in the whole culture period, while the effect of LaeA mainly occurred in the later phases of prolonged batch cultures. Similar to LaeA, Mtr23B was involved in the expression of 10 physically linked regions containing secondary metabolic gene clusters; the highest regulatory activities of Mtr23B and LaeA were observed in BrlA-dependent cascades. Although LaeA interacted with VeA, Mtr23B did not interact with VeA directly. We assumed that Mtr23B regulates cellulase and amylase gene transcription by interacting with the CCAAT-binding transcription factor HAP5 and chromatin remodeling complex.

This paper discusses the evaluation and prototyping of a 12V/3.3V Gallium-Nitride (GaN) point-of-load converter for satellite applications. Traditional power converters used in spacecraft are designed with silicon devices, and there are limitations in efficiency, package parasitic, and thermal management that must be addressed at frequencies above 500kHz. GaN HEMTs have advantages of TID tolerance, shorter turn-on/off time, and lower driving loss. By utilizing GaN HEMTs, switching frequency of 2MHz is selected to trade off volume and efficiency. Low parasitic loop inductance is realized by appropriate layout, and the peak voltage of the switches has been reduced. With the 3-D integration architecture, the junction-to-board thermal resistance has been reduced, and 20A output current is realized in a volume of $15\times 15\times 5.5\mathrm{mm}^{3}$ . Calculations, simulations and experimental results are provided to evaluate the performance of the converter.

Numerical investigation of dielectric barrier discharge (DBD) plasma actuation has been conducted to quantify the momentum transfer without and with the incoming flow over a flat plate. A comparative analysis between numerical investigations and experiments is presented. A phenomenological model is used to simplify the effect of plasma on air flow based on the momentum balance equation. For steady actuation, the plasma will produce a steady jet close to the wall, the effect region of plasma is gradually getting larger and the maximum velocity of the jet is reducing along the chord of the actuator. The main way of flow control is to inject momentum into incoming flow, and the induced jet is continuously transferred with nearby fluid. For unsteady actuation, the plasma will produce a series of vortex structures and the intensity of them decreases gradually along the chord of plasma actuator. The mechanism of unsteady plasma actuation is both the momentum and vorticity injection.

Additional file 9: Table S2. Primers used in this study.

Some Penicillium species have been reported to produce enzyme systems with good performances in lignocellulose degradation. Penicillium oxalicum (formerly classified as P. decumbens) strains, which produce more balanced native lignocellulolytic enzyme systems than Trichoderma reesei, have been studied for more than 30 years. The original P. oxalicum isolate 114 was obtained from decayed straw-covered soil in 1979 and has been improved through a series of mutagenesis and screening over the years. The whole genome sequence of the P. oxalicum was finished in 2009. Comparative and functional genomics studies between P. oxalicum mutant JU-A10-T and wild-type strain 114-2 were performed to decipher how strain improvement has significantly improved the production of the lignocellulolytic enzyme system. Further, the transcriptomes and secretomes of the P. oxalicum were determined. The applications of genomic, transcriptomic, and proteomic analysis methods make it possible in evaluation of the native enzyme system, discovery of novel auxiliary proteins, understanding of regulatory mechanisms by key transcription factors, and exploration of the cellular network controlling lignocellulolytic enzyme synthesis. A single-gene disruptant library for 470 transcription factors was constructed, and several activators and repressors were identified to play essential roles in regulating lignocellulolytic enzyme production. Redesigning the regulatory pathway substantially improves lignocellulolytic enzyme production up to the industrial level by combinational manipulation of three key genes to amplify the induction along with derepression. By combining systems biology tools, engineered fungal strains are expected to produce high levels of optimized lignocellulolytic enzyme systems.

The filamentous fungus, Beauveria bassiana, is a ubiquitously distributed insect pathogen, currently used as an alternative to chemical pesticides for pest control. Conidiospores are the means by which the fungus disseminates in the environment, and these cells also represent the infectious agent most commonly used in field applications. Little, however, is known concerning the molecular basis for maintenance of spore viability, a critical feature for survival and persistence. Here, we report on the role of a putative methyltransferase, BbmtrA, in conidial viability, normal fungal growth and development, and virulence, via characterization of a targeted gene knockout strain. Loss of BbmtrA resulted in pleiotropic effects including reduced germination, growth and conidiation, with growing mycelia displaying greater branching than the WT parent. Conidial viability dramatically decreased over time, with 80 % of the WT. Reduced production of extracellular proteins was also observed for the ΔBbmtrA mutant, including protease/peptidases, glycoside hydrolases and the hyd1 hydrophobin. The latter was further confirmed by hyd1 gene expression analysis. Insect bioassays using the greater wax moth, Galleria mellonella, further revealed that the ΔBbmtrA strain was attenuated in virulence and failed to sporulate on host cadavers. These data support a global role for mtrA in fungal physiological processes.

SummaryBrain circuits endow behavioral flexibility. Here, we study circuits encoding flexible chemotaxis in C. elegans, where the animal navigates up or down NaCl gradients (positive or negative chemotaxis) to reach the salt concentration of previous growth (the set point). The ASER sensory neuron mediates positive and negative chemotaxis by regulating the frequency and direction of reorientation movements in response to salt gradients. Both salt gradients and set point memory are encoded in ASER temporal activity patterns. Distinct temporal activity patterns in interneurons immediately downstream of ASER encode chemotactic movement decisions. Different interneuron combinations regulate positive versus negative chemotaxis. We conclude that sensorimotor pathways are segregated immediately after the primary sensory neuron in the chemotaxis circuit, and sensory representation is rapidly transformed to motor representation at the first interneuron layer. Our study reveals compact encoding of perception, memory, and locomotion in an experience-dependent navigational behavior in C. elegans.

Casein kinase CK2 is a ubiquitous and conserved phosphate transferase that is critical for the growth and development of eukaryotic cells. In Penicillium oxalicum, one catalytic subunit (CK2A) and two regulatory subunits (CK2B1 and CK2B2) of CK2 were annotated. In this study, CK2 regulatory subunit-defective mutants Δck2B1 and Δck2B2 were constructed to investigate the biological function of CK2 in P. oxalicum. The Δck2B1 strain exhibited minimal changes in morphogenesis and conidiation, whereas the Δck2B2 strain showed delayed conidial germination and drastically reduced conidiation compared with the parent strain. The defect in conidiation in Δck2B2 could be attributed to the reduced expression of transcription factor BrlA. Both Δck2B1 and Δck2B2 showed delayed autolysis in carbon-starvation medium compared with the parent strain. Cellulase and amylase production were decreased considerably in both mutants. The transcript abundances of the main extracellular glycoside hydrolase genes cel7A-2, bgl1, and amy15A, as well as those of three related transcriptional activators (i.e., ClrB, XlnR, and AmyR), were reduced or delayed in the mutants. Epistasis analysis suggested that CK2B1 and CK2B2 might function upstream of transcription factor CreA by inhibiting its repressing activity. In summary, CK2 plays important roles in development and extracellular enzyme production in P. oxalicum, with both unique and overlapping functions performed by the two regulatory subunits.

α-l-Arabinofuranosidases are important in the degradation of plant polysaccharides and are used in several industrial processes. Although the use of filamentous fungi for the production of α-l-arabinofuranosidases is widely reported, there are few reports on strain engineering for enhanced production of these enzymes by fungi. In this study, the function of transcription factor AraR in l-arabinose release and catabolism by the fungus Penicillium oxalicum (P. oxalicum) is investigated. Also, a mutant of AraR, AraRA731V , is constructed to improve the production of α-l-arabinofuranosidases on the basis of the sequence homology between AraR and the xylanolytic gene activator XlnR. The AraRA731V -overexpressing strain can synthesize α-l-arabinofuranosidase in the absence of an inducer and shows a 54.1-fold increase in α-l-arabinofuranosidase production and a 7.4-fold increase in α-galactosidase production in the medium containing wheat bran. Determination of the transcript abundances of lignocellulolytic enzyme genes reveals significant upregulation of multiple α-l-arabinofuranosidase genes and downregulation of some cellulolytic and xylanolytic enzyme genes in the engineered strain relative to its parent. Taken together, the results suggest the conserved regulatory function of AraR in the family Trichocomaceae and provide a strategy for engineering fungal strains for enhanced α- l-arabinofuranosidase production.

Summary For most organisms, carbon and nitrogen uptake are essential for growth, development and, where applicable, pathogenesis. The role of the carbon catabolite repressor transcription factor homologue BbcreA in the entomopathogenic fungus Beauveria bassiana was investigated. Deletion of BbcreA resulted in pleiotropic effects, including nutrient toxicity, leading to a novel cell lytic phenotype. Fungal growth in rich media and minimal media containing select amino acids/peptides was severely compromised, with microscopic examination revealing conidial-base germ tube degeneration and cell lysis occurring during growth, a phenomenon exacerbated at higher temperatures (32°C). Depending upon nutrient conditions, growth, pigment and aerial mycelium production, sporulation and dimorphic transition to blastospore production were also impaired in the ΔBbcreA strain. Although loss of BbcreA resulted in de-repression of secreted protease and lipase, enzymes critical in mediating pathogenesis, insect bioassays indicated severe defects in virulence using both topical and intra-haemocoel injection assays, with eruption and subsequent sporulation on host cadavers greatly reduced in the mutant. These data suggest that BbcreA functions as more than a carbon repressor and plays important roles in nutrient utilization, cell homeostasis and virulence. In particular, BbcreA is required for proper assimilation of select amino acids and peptides, including asparagine, arginine and proline.

The current high cost of lignocellulolytic enzymes is a major bottleneck in the economic bioconversion of lignocellulosic biomass to fuels and chemicals. Fungal lignocellulolytic enzyme systems are secreted at high levels, making them the most promising starting points for further development of highly efficient lignocellulolytic enzyme systems. In this paper, recent advances in improvement of fungal lignocellulolytic enzyme systems are reviewed, with an emphasis on the achievements made using genomic approaches. A general strategy for lignocellulolytic enzyme system development is proposed, including the improvement of the hydrolysis efficiencies and productivities of current enzyme systems. The applications of genomic, transcriptomic and proteomic analysis methods in examining the composition of native enzyme systems, discovery of novel enzymes and synergistic proteins from natural sources, and understanding of regulatory mechanisms for lignocellulolytic enzyme biosynthesis are summarized. By combining systems biology and synthetic biology tools, engineered fungal strains are expected to produce high levels of optimized lignocellulolytic enzyme systems.

Cellodextrin transporters (cellodextrin permeases) have been identified in fungi in recent years. However, the functions of these transporters in cellulose utilization and cellulase expression have not been well studied. In this study, three cellodextrin transporters, namely, CdtC, CdtD, and CdtG, in the cellulolytic fungus Penicillium oxalicum (formally was classified as P. decumbens) were identified, and their functions were analyzed. The deletion of a single cellodextrin transporter gene slightly decreased cellobiose consumption, but no observable effect on cellulase expression was observed, which was attributed to the overlapping activity of isozymes. Further simultaneous deletion of cdtC and cdtD resulted in significantly decreased cellobiose consumption and poor growth on cellulose. The extracellular activity and transcription level of cellulases in the mutant without cdtC and cdtD were significantly lower than those in the wild-type strain when grown on cellulose. This result provides direct evidence of the crucial function of cellodextrin transporters in the induction of cellulase expression by insoluble cellulose.