VIB1 is not required for cellobiose sensing or signaling.

<p>(A) Expression levels of two cellulase genes (<i>cbh-1</i> and <i>gh5-1</i>) were assessed in the Δ3βG; <i>Δvib-1</i> strain versus WT, and the Δ3βG and <i>Δvib-1</i> strains after a shift from sucrose VMM to 0.2% cellobiose versus 2% Avicel. Gene expression levels were measured by relative quantitative-PCR using actin as a control and normalized to expression level when cultures were shifted to VMM with no carbon source. (B) CMCase activity after 24 hrs of growth on 2% cellobiose of the Δ3βG; <i>Δvib-1</i> strain relative to the WT, and the Δ3βG and <i>Δvib-1</i> strains. Measured enzyme activity in arbitrary unit (AU) was normalized against to the mycelial biomass of each culture.</p

Suppression of <i>cre-1</i> and <i>col-26</i> expression by VIB1 plays a role in early inductive and utilization phase during growth on cellulose.

<p>The transcriptional expression of <i>cre-1</i>, <i>vib-1</i>, <i>col-26</i>, <i>clr-2</i>, and <i>cbh-1</i> were measured by RT-PCR at 4 hrs (A) and 24 hrs (B) after 16 hr sucrose growth cultures were transferred to Avicel conditions. Expression levels were normalized to WT.</p

Cellulase production in <i>N. crassa</i> is regulated by cellobiose induction and CCR.

<p>CCR is decreased in absence of glucose, allowing scouting enzymes to liberate cellobiose from cellulose. Cellobiose (or a derivative) results in activation of the transcription factor CLR1, which induces expression of transporters for cellodextrins, β-glucosidases, and <i>clr-2</i>. Production of the transcription factor CLR2 drives cellulase gene expression. Both intracellular and extracellular β-glucosidase enzymes catalyze conversion of cellobiose to glucose, which can trigger carbon catabolite repression via glucose sensing mechanisms and transcriptional repression by CRE1.</p

Screen for function of new proteins involved in CCR.

<p>(A) Growth assays of the WT, <i>Δvib-1</i>, Δ<i>cre-1</i>, Δ<i>col-26</i>, Δ<i>creB</i>, and Δ<i>creD</i> strains on 2-deoxy-glucose (2-DG) when grown on 2% cellobiose VMM for 2 days or on 2% Avicel VMM for 4 days. (B) Effects of <i>col-26</i> and <i>cre-1</i> deletions on sensitivity to 2-DG and allyl alcohol. Strains were inoculated and grown in 2% cellobiose VMM with 100 mM allyl alcohol for 40 hrs. For 2-DG sensitivity tests, the strains were inoculated and grown in 2% Avicel with either 0.2% 2-DG or 0.5% 2-DG for 5 days.</p

Constitutive expression of <i>clr-2</i> rescued the cellulase production defect of the Δ<i>vib-1</i> mutant.

<p>(A) Protein concentration and cellulase activity in a Δ<i>vib-1</i> mutant versus a Δ<i>vib-1</i> strain constitutively expressing <i>clr-2</i> (Pc <i>clr-2</i>; Δ<i>vib-1</i>) and WT and a Pc <i>clr-2</i> strain under Avicel conditions. (B) Expression levels from RNA-seq data of genes encoding major classes of CAZy proteins from WT and Δ<i>vib-1</i> shifted to Avicel versus the Pc <i>clr-2</i> and Pc <i>clr-2</i>; Δ<i>vib-</i>1 strains shifted to minimal media with no carbon source. FPKM (Fragment Per Kilobase per exon per Megabase mapped) for individual genes were averaged between three biological replicates and pooled by CAZy class. (C) Hierarchical clustering of FPKM for 91 Avicel-regulon genes in the Δ<i>vib-1</i> mutant and WT on Avicel (Av) and the Δ<i>vib-1</i>, WT, Pc <i>clr-2</i> and the Pc <i>clr-2</i>; Δ<i>vib-</i>1 strains switched to no carbon conditions (Nc). Results are displayed as heat maps with log (FPKM) from minimum (bright blue) to maximum (bright yellow).</p

Deletion of <i>vib-1</i> abolishes production of cellulases and utilization of cellulosic material.

<p>(A) Growth of WT and Δ<i>vib-1</i> on Avicel after 4 days; growth of WT is indicated by formation of orange mycelia, versus no growth of the Δ<i>vib-1</i> mutant. (B) Cellulase activity from 4-day old culture supernatants from Avicel-grown cultures of WT, the Δ<i>vib-1</i> mutant, the P<i>vib-1</i> strain (constitutive expression of <i>vib-1</i> in a Δ<i>vib-1</i> strain) and the P<i>Trvib1</i> strain (constitutive expression of <i>T. reesei vib1</i> in a Δ<i>vib-1</i> strain). Cellulase activity was measured using Avicel as a substrate and represented by the amount of glucose and cellobiose released. The equivalent of glucose from cellobiose was calculated and represented by the light gray bar. (C) The secretomes of strains analyzed in panel B are shown.</p

Simultaneous deletion of <i>cre-1</i> and <i>col-26</i> rescues the phenotype of Δ<i>vib-1</i> on cellulose.

<p>(A) Cellulase activity of culture supernatants after 4-days of growth on Avicel from WT versus Δ<i>vib-1</i>, Δ<i>cre-1</i>, Δ<i>col-26</i>, Δ<i>cre-1</i>; Δ<i>vib-1</i>, Δ<i>col-26</i>; Δ<i>vib-1</i> and Δ<i>cre-1</i>; Δ<i>col-26</i>; Δ<i>vib-1</i> strains. (B) RT-PCR measurements of <i>clr-2</i> and <i>cbh-1</i> expression in the WT versus the Δ<i>col-26</i>, Δ<i>cre-1</i>, and the Δ<i>cre-1</i>; Δ<i>col-26</i>; Δ<i>vib-1</i> cultures after 5-days of growth on Avicel. Expression levels were normalized to WT. (C) The CMCase activity of Avicel cultures of WT versus Δ<i>col-26</i>, Δ<i>cre-1</i>, and the Δ<i>cre-1</i>; Δ<i>col-26</i>; Δ<i>vib-1</i> mutants during a time course of growth on Avicel.</p

Fig 10 -

(a) The minimum sound power level of multi-working modification parameter under continuous working conditions. (b) The minimum sound power level of multi-working modification parameter under high-speed conditions.</p

Gear set transmission linearity error data.

The vibration and radiation noise characteristics of the gear transmission system are different under different traction conditions, and the gear modification optimization scheme based on a single working condition is not suitable for the operating environment under all working conditions. To modify the traction gear of a high-speed EMU, an optimized design scheme for noise reduction under multiple working conditions is proposed. A modification plan of the tooth direction in conjunction with the tooth shape was devised using a parametric model of the EMU’s traction gear transmission system. The radiation noise of the gear transmission system after modification was solved using the acoustic boundary element method under different working conditions. A gear noise prediction model based on the random forest was proposed, and a gear modification parameter combination was constructed to minimize radiation noise. Then, the optimal design scheme of multi-condition modification combination parameters is obtained with the weight of the running time and acoustic contribution under different working conditions. The grey correlation degree evaluation model is established to verify that the multi-condition modification optimization design method can make the traction gear of EMU obtain satisfactory transmission performance and noise reduction effect under different working conditions.</div

VIB1, a Link between Glucose Signaling and Carbon Catabolite Repression, Is Essential for Plant Cell Wall Degradation by <i>Neurospora crassa</i>

<div><p>Filamentous fungi that thrive on plant biomass are the major producers of hydrolytic enzymes used to decompose lignocellulose for biofuel production. Although induction of cellulases is regulated at the transcriptional level, how filamentous fungi sense and signal carbon-limited conditions to coordinate cell metabolism and regulate cellulolytic enzyme production is not well characterized. By screening a transcription factor deletion set in the filamentous fungus <i>Neurospora crassa</i> for mutants unable to grow on cellulosic materials, we identified a role for the transcription factor, VIB1, as essential for cellulose utilization. VIB1 does not directly regulate hydrolytic enzyme gene expression or function in cellulosic inducer signaling/processing, but affects the expression level of an essential regulator of hydrolytic enzyme genes, CLR2. Transcriptional profiling of a Δ<i>vib-1</i> mutant suggests that it has an improper expression of genes functioning in metabolism and energy and a deregulation of carbon catabolite repression (CCR). By characterizing new genes, we demonstrate that the transcription factor, COL26, is critical for intracellular glucose sensing/metabolism and plays a role in CCR by negatively regulating <i>cre-1</i> expression. Deletion of the major player in CCR, <i>cre-1</i>, or a deletion of <i>col-26</i>, did not rescue the growth of Δ<i>vib-1</i> on cellulose. However, the synergistic effect of the Δ<i>cre-1</i>; Δ<i>col-26</i> mutations circumvented the requirement of VIB1 for cellulase gene expression, enzyme secretion and cellulose deconstruction. Our findings support a function of VIB1 in repressing both glucose signaling and CCR under carbon-limited conditions, thus enabling a proper cellular response for plant biomass deconstruction and utilization.</p></div