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.

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.

No abstract

To increase the number of value-added chemicals that can be produced by metabolic engineering and synthetic biology, constructing metabolic space with novel reactions/pathways is crucial. However, with the large number of reactions that existed in the metabolic space and complicated metabolisms within hosts, identifying novel pathways linking two molecules or heterologous pathways when engineering a host to produce a target molecule is an arduous task. Hence, we built a user-friendly web server, novoPathFinder, which has several features: (i) enumerate novel pathways between two specified molecules without considering hosts; (ii) construct heterologous pathways with known or putative reactions for producing target molecule within Escherichia coli or yeast without giving precursor; (iii) estimate novel pathways with considering several categories, including enzyme promiscuity, Synthetic Complex Score (SCScore) and LD50 of intermediates, overall stoichiometric conversions, pathway length, theoretical yields and thermodynamic feasibility. According to the results, novoPathFinder is more capable to recover experimentally validated pathways when comparing other rule-based web server tools. Besides, more efficient pathways with novel reactions could also be retrieved for further experimental exploration. novoPathFinder is available at http://design.rxnfinder.org/novopathfinder/.

No abstract