Direct C–H Trifluoromethylation of Glycals by Photoredox Catalysis

A mild, efficient, and practical transformation for the direct C–H trifluoromethylation of glycals under visible light has been reported for the first time. This reaction employed fac-Ir³⁺(ppy)₃ as the photocatalyst, Umemoto’s reagent as the CF₃ source, and a household blue LED or sunlight as the light source. Glycals bearing both electron-withdrawing and -donating protective groups performed this reaction smoothly. This visible light-mediated trifluoromethylation reaction was highlighted by the trifluoromethylation of the biologically important Neu2en moiety

Evidence of a Critical Role for Cellodextrin Transporte 2 (CDT-2) in Both Cellulose and Hemicellulose Degradation and Utilization in <i>Neurospora crassa</i>

<div><p>CDT-1 and CDT-2 are two cellodextrin transporters discovered in the filamentous fungus <i>Neurospora crassa</i>. Previous studies focused on characterizing the role of these transporters in only a few conditions, including cellulose degradation, and the function of these two transporters is not yet completely understood. In this study, we show that deletion of <i>cdt-2</i>, but not <i>cdt-1</i>, results in growth defects not only on Avicel but also on xylan. <i>cdt-2</i> can be highly induced by xylan, and this mutant has a xylodextrin consumption defect. Transcriptomic analysis of the <i>cdt-2</i> deletion strain on Avicel and xylan showed that major cellulase and hemicellulase genes were significantly down-regulated in the <i>cdt-2</i> deletion strain and artificial over expression of <i>cdt-2</i> in <i>N. crassa</i> increased cellulase and hemicellulase production. Together, these data clearly show that CDT-2 plays a critical role in hemicellulose sensing and utilization. This is the first time a sugar transporter has been assigned a function in the hemicellulose degradation pathway. Furthermore, we found that the transcription factor XLR-1 is the major regulator of <i>cdt-2</i>, while <i>cdt-1</i> is primarily regulated by CLR-1. These results deepen our understanding of the functions of both cellodextrin transporters, particularly for CDT-2. Our study also provides novel insight into the mechanisms for hemicellulose sensing and utilization in <i>N. crassa</i>, and may be applicable to other cellulolytic filamentous fungi.</p></div

Xylobiose and xylotriose consumption by WT, Δ<i>cdt-2</i> and Pc-<i>cdt-2</i>.

<p>All strains were grown in liquid MM media for 16% xylan or 0.5% sucrose. Ten milliliters of mycelia were collected and incubated with 90 µM of either xylobiose or xylotriose for 15 min. The mean and deviation represent three independent measurements. *<i>P</i><0.05 (two sided student’s t-test).</p

Venn diagram of the transcriptome comparison of WT and Δ<i>cdt-2</i> on either Avicel or xylan.

<p>A) The genes that show a statistically significant down-regulation in expression level in Δ<i>cdt-2</i> compared with WT on xylan or Avicel. Fourteen genes that were down-regulated on both Avicel and xylan in the Δ<i>cdt-2</i> strain compared with WT are related to polysaccharide metabolism (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089330#pone-0089330-t002" target="_blank">Table 2</a>). B) The genes that show a statistically significant up-regulation in expression level in Δ<i>cdt-2</i> compared with WT on xylan or Avicel. Funcat analyses of all gene sets are available in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089330#pone.0089330.s002" target="_blank">Table S2</a>. The wild type and Δ<i>cdt-2</i> strains were grown in liquid MM media for 16 h, then transferred into 0.5% xylan or 0.5% Avicel for an additional 4 h of cultivation.</p

Relative expression levels of <i>cdt-2</i> and <i>cdt-1</i> in different mutants determined by qRT-PCR.

<p>The mutants carrying deletions for different transcription factors (Δ<i>xlr-1</i>, Δ<i>clr-1</i>, Δ<i>clr-2</i> and Δ<i>cre-1</i>) were grown in liquid MM media for 16 h, then transferred into 0.5% xylan or 0.5% Avicel for an additional 4 h of cultivation. Gene expression levels of actin (NCU04173) were used as an endogenous control in all samples. Each reaction was done by triplicate. *<i>P</i><0.05 (two sided student’s t-test).</p

Gene expression level of <i>cdt-1</i> and <i>cdt-2</i> on different carbon sources.

<p>Gene expression levels of <i>cdt-1</i> and <i>cdt-2</i> on different carbon sources (glucose, cellobiose, Avicel, xylose, xylodextrin and xylan) by qRT-PCR. The wild type strains were grown on different carbon sources at 25°C for 16 h (glucose, cellobiose, xylodextrin and xylan), 22 h (xylose), or 30 h (Avicel). Gene expression levels of actin (NCU04173) were used as an endogenous control in all samples. Each reaction was done in triplicate.</p

Enzyme activity of culture supernatants of wild type and CPL-1 strains.

<p>A) β-xylosidase activity and xylanase activity of wild type strain and <i>cdt-2</i> overexpression strain CPL-1 on xylan for 1, 3 and 5 days of cultivation. B) Endoglucanase activity and xylanase activity of WT and CPL-1 on Avicel for 1, 3 and 5 days of cultivation. The mean and deviation showed are three independent measurements. *<i>P</i><0.05 (two sided student’s t-test).</p

Microscopic observation of CDT-2 subcellular localization in <i>N. crassa</i>.

<p>Pc-<i>cdt-2</i> and Pn-<i>cdt-2</i> were grown in liquid MM media for 16 h and transferred into inducing media with Vogel’s salts and either 0.5% xylan or 0.5% Avicel as carbon source at 25°C for an additional 4 h of cultivation. Microscopic observation was performed by a Laser Scanning Confocal Microscope (Leica TCS SP5 II). The nuclei were stained by DAPI. Scale bar = 10 µm.</p

Strains and plasmids used in this study.

<p>Strains and plasmids used in this study.</p

Down-regulated genes related to polysaccharide metabolism in Δ<i>cdt-2</i> strain compared with WT on both Avicel and xylan media.

a<p>GPI-anchored cell wall β-1,3-endoglucanase EglC.</p>b<p>Avicel.</p>c<p>xylan.</p