BackgroundThe glucose dual-affinity transport system (low- and high-affinity) is a conserved strategy used by microorganisms to cope with natural fluctuations in nutrient availability in the environment. The glucose-sensing and uptake processes are believed to be tightly associated with cellulase expression regulation in cellulolytic fungi. However, both the identities and functions of the major molecular components of this evolutionarily conserved system in filamentous fungi remain elusive. Here, we systematically identified and characterized the components of the glucose dual-affinity transport system in the model fungus Neurospora crassa.ResultsUsing RNA sequencing coupled with functional transport analyses, we assigned GLT-1 (K m = 18.42 ± 3.38 mM) and HGT-1/-2 (K m = 16.13 ± 0.95 and 98.97 ± 22.02 µM) to the low- and high-affinity glucose transport systems, respectively. The high-affinity transporters hgt-1/-2 complemented a moderate growth defect under high glucose when glt-1 was deleted. Simultaneous deletion of hgt-1/-2 led to extensive derepression of genes for plant cell wall deconstruction in cells grown on cellulose. The suppression by HGT-1/-2 was connected to both carbon catabolite repression (CCR) and the cyclic adenosine monophosphate-protein kinase A pathway. Alteration of a residue conserved across taxa in hexose transporters resulted in a loss of glucose-transporting function, whereas CCR signal transduction was retained, indicating dual functions for HGT-1/-2 as “transceptors.”ConclusionsIn this study, GLT-1 and HGT-1/-2 were identified as the key components of the glucose dual-affinity transport system, which plays diverse roles in glucose transport and carbon metabolism. Given the wide conservation of the glucose dual-affinity transport system across fungal species, the identification of its components and their pleiotropic roles in this study shed important new light on the molecular basis of nutrient transport, signaling, and plant cell wall degradation in fungi.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-017-0705-4) contains supplementary material, which is available to authorized users.

CDT-1 and CDT-2 are two cellodextrin transporters discovered in the filamentous fungus Neurospora crassa. 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 cdt-2, but not cdt-1, results in growth defects not only on Avicel but also on xylan. cdt-2 can be highly induced by xylan, and this mutant has a xylodextrin consumption defect. Transcriptomic analysis of the cdt-2 deletion strain on Avicel and xylan showed that major cellulase and hemicellulase genes were significantly down-regulated in the cdt-2 deletion strain and artificial over expression of cdt-2 in N. crassa 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 cdt-2, while cdt-1 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 N. crassa, and may be applicable to other cellulolytic filamentous fungi.

CRISPR-Staphylococcus aureus Cas9 (CRISPR-SaCas9) has been harnessed as an effective in vivo genome-editing tool to manipulate genomes. However, off-target effects remain a major bottleneck that precludes safe and reliable applications in genome editing. Here, we characterize the off-target effects of wild-type (WT) SaCas9 at single-nucleotide (single-nt) resolution and describe a directional screening system to identify novel SaCas9 variants with desired properties in human cells. Using this system, we identified enhanced-fidelity SaCas9 (efSaCas9) (variant Mut268 harboring the single mutation of N260D), which could effectively distinguish and reject single base-pair mismatches. We demonstrate dramatically reduced off-target effects (approximately 2-to 93-fold improvements) of Mut268 compared to WT using targeted deep-sequencing analyses. To understand the structural origin of the fidelity enhancement, we find that N260, located in the REC3 domain, orchestrates an extensive network of contacts between REC3 and the guide RNA-DNA heteroduplex. efSa-Cas9 can be broadly used in genome-editing applications that require high fidelity. Furthermore, this study provides a general strategy to rapidly evolve other desired CRISPR-Cas9 traits besides enhanced fidelity, to expand the utility of the CRISPR toolkit.

Background: The components of the cellulase induction pathway in fungi remain unclear. Results: Identify the hypothetical protein CLP1 negatively regulates the cellulases induction through working with cellodextrin transporters CDT1 and CDT2. Conclusion: CLP1 is a novel element of the cellulase induction pathway. Significant: These data deepen the understanding of cellulase induction pathway and provide a new strategy to improve fungal cellulase production.

cLimited uptake is one of the bottlenecks for L-arabinose fermentation from lignocellulosic hydrolysates in engineered Saccharomyces cerevisiae. This study characterized two novel L-arabinose transporters, LAT-1 from Neurospora crassa and MtLAT-1 from Myceliophthora thermophila. Although the two proteins share high identity (about 83%), they display different substrate specificities. Sugar transport assays using the S. cerevisiae strain EBY.VW4000 indicated that LAT-1 accepts a broad substrate spectrum. In contrast, MtLAT-1 appeared much more specific for L-arabinose. Determination of the kinetic properties of both transporters revealed that the K m values of LAT-1 and MtLAT-1 for L-arabinose were 58.12 ؎ 4.06 mM and 29.39 ؎ 3.60 mM, respectively, with corresponding V max values of 116.7 ؎ 3.0 mmol/h/g dry cell weight (DCW) and 10.29 ؎ 0.35 mmol/h/g DCW, respectively. In addition, both transporters were found to use a proton-coupled symport mechanism and showed only partial inhibition by D-glucose during L-arabinose uptake. Moreover, LAT-1 and MtLAT-1 were expressed in the S. cerevisiae strain BSW2AP containing an L-arabinose metabolic pathway. Both recombinant strains exhibited much faster L-arabinose utilization, greater biomass accumulation, and higher ethanol production than the control strain. In conclusion, because of higher maximum velocities and reduced inhibition by D-glucose, the genes for the two characterized transporters are promising targets for improved L-arabinose utilization and fermentation in S. cerevisiae. Biorefining of lignocellulosic biomass has attracted considerable attention in recent years because of its abundance, sustainability and potential environmental benefits (1, 2). The main sugars in hydrolysates from currently used feedstocks are a mixture of D-glucose, D-xylose, and L-arabinose (3). For a cost-effective conversion of biomass into fuels or chemicals, the fermentative organisms are required to utilize all three sugars efficiently. Even though many organisms are able to natively convert these substrates, the most commonly selected microbe is Saccharomyces cerevisiae (baker's yeast) because of its high ethanol productivity and high tolerance for inhibitors present in biomass hydrolysates, as well as the fact that it is amenable to genetic engineering (4-6).Wild-type S. cerevisiae cannot utilize the two pentose sugars D-xylose and L-arabinose. Thus, improving import and the intracellular pentose utilization efficiency is very critical, and intensive efforts have been made to do so by yeast metabolic engineering (7-13). Sugar uptake is the initial step for its utilization, and therefore, efficient molecular transport is a prerequisite to achieve enhanced fermentation rates in the presence of a working pentose metabolism pathway. Baker's yeast is able to absorb pentoses through its native hexose transporters, such as Hxt5 and Hxt7, and the galactose transporter Gal2 (14-16). However, these transporters show higher affinity for hexoses, and thus, it was suggested that D-glucose impa...

BackgroundCrop residue is an abundant, low-cost plant biomass material available worldwide for use in the microbial production of enzymes, biofuels, and valuable chemicals. However, the diverse chemical composition and complex structure of crop residues are more challenging for efficient degradation by microbes than are homogeneous polysaccharides. In this study, the transcriptional responses of Neurospora crassa to various plant straws were analyzed using RNA-Seq, and novel beneficial factors for biomass-induced enzyme production were evaluated.ResultsComparative transcriptional profiling of N. crassa grown on five major crop straws of China (barley, corn, rice, soybean, and wheat straws) revealed a highly overlapping group of 430 genes, the biomass commonly induced core set (BICS). A large proportion of induced carbohydrate-active enzyme (CAZy) genes (82 out of 113) were also conserved across the five plant straws. Excluding 178 genes within the BICS that were also upregulated under no-carbon conditions, the remaining 252 genes were defined as the biomass regulon (BR). Interestingly, 88 genes were only induced by plant biomass and not by three individual polysaccharides (Avicel, xylan, and pectin); these were denoted as the biomass unique set (BUS). Deletion of one BUS gene, the transcriptional regulator rca-1, significantly improved lignocellulase production using plant biomass as the sole carbon source, possibly functioning via de-repression of the regulator clr-2. Thus, this result suggests that rca-1 is a potential engineering target for biorefineries, especially for plant biomass direct microbial conversion processes.ConclusionsTranscriptional profiling revealed a large core response to different sources of plant biomass in N. crassa. The sporulation regulator rca-1 was identified as beneficial for biomass-based enzyme production.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-015-0208-0) contains supplementary material, which is available to authorized users.

BackgroundGene gain and loss frequently occurs in the cyanobacterium Prochlorococcus, a phototroph that numerically dominates tropical and subtropical open oceans. However, little is known about the stabilization of its core genome, which contains approximately 1250 genes, in the context of genome streamlining. Using Prochlorococcus MED4 as a model organism, we investigated the constraints on core genome stabilization using transcriptome profiling.ResultsRNA-Seq technique was used to obtain the transcriptome map of Prochlorococcus MED4, including operons, untranslated regions, non-coding RNAs, and novel genes. Genome-wide expression profiles revealed that three factors contribute to core genome stabilization. First, a negative correlation between gene expression levels and protein evolutionary rates was observed. Highly expressed genes were overrepresented in the core genome but not in the flexible genome. Gene necessity was determined as a second powerful constraint on genome evolution through functional enrichment analysis. Third, quick mRNA turnover may increase corresponding proteins’ fidelity among genes that were abundantly expressed. Together, these factors influence core genome stabilization during MED4 genome evolution.ConclusionsGene expression, gene necessity, and mRNA turnover contribute to core genome maintenance during cyanobacterium Prochlorococcus genus evolution.

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