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Background Cellulose is not only a common component in vascular plants, but also has great economic benefits for paper, wood, and industrial products. In addition, its biosynthesis is highly regulated by carbohydrate metabolism and allocation in plant. MdFRK2, which encodes a key fructokinase (FRK) in apple, showed especially high affinity to fructose and regulated carbohydrate metabolism. Results It was observed that overexpression of MdFRK2 in apple decreased sucrose (Suc) and fructose (Fru) with augmented FRK activity in stems, and caused the alterations of many phenotypic traits that include increased cellulose content and an increase in thickness of the phloem region. To further investigate the involved mechanisms, we generated FRK2-OE poplar lines OE#1, OE#4 and OE#9 and discovered (1) that overexpression of MdFRK2 resulted in the huge increased cellulose level by shifting the fructose 6-phosphate or glucose 6-phsophate towards UDPG formation, (2) a direct metabolic pathway for the biosynthesis of cellulose is that increased cleavage of Suc into UDP-glucose (UDPG) for cellulose synthesis via the increased sucrose synthase (SUSY) activity and transcript levels of PtrSUSY1, (3) that the increased FRK activity increases the sink strength overall so there is more carbohydrate available to fuel increased cambial activity and that resulted in more secondary phloem. These results demonstrated that MdFRK2 overexpression would significantly changes the photosynthetic carbon flux from sucrose and hexose to UDPG for increased cellulose synthesis. Conclusions The present data indicated that MdFRK2 overexpression in apple and poplar changes the photosynthetic carbon flux from sucrose and hexose to UDPG for stem cellulose synthesis. A strategy is proposed to increase cellulose production by regulating sugar metabolism as a whole.

Background Caldicellulosiruptor kronotskyensis has gained interest for its ability to grow on various lignocellulosic biomass. The aim of this study was to investigate the growth profiles of C. kronotskyensis in the presence of mixtures of glucose–xylose. Recently, we characterized a diauxic-like pattern for C. saccharolyticus on lignocellulosic sugar mixtures. In this study, we aimed to investigate further whether C. kronotskyensis has adapted to uptake glucose in the disaccharide form (cellobiose) rather than the monosaccharide (glucose). Results Interestingly, growth of C. kronotskyensis on glucose and xylose mixtures did not display diauxic-like growth patterns. Closer investigation revealed that, in contrast to C. saccharolyticus, C. kronotskyensis does not possess a second uptake system for glucose. Both C. saccharolyticus and C. kronotskyensis share the characteristics of preferring xylose over glucose. Growth on xylose was twice as fast (μmax = 0.57 h−1) as on glucose (μmax = 0.28 h−1). A study of the sugar uptake was made with different glucose–xylose ratios to find a kinetic relationship between the two sugars for transport into the cell. High concentrations of glucose inhibited xylose uptake and vice versa. The inhibition constants were estimated to be KI,glu = 0.01 cmol L−1 and KI,xyl = 0.001 cmol L−1, hence glucose uptake was more severely inhibited by xylose uptake. Bioinformatics analysis could not exclude that C. kronotskyensis possesses more than one transporter for glucose. As a next step it was investigated whether glucose uptake by C. kronotskyensis improved in the form of cellobiose. Indeed, cellobiose is taken up faster than glucose; nevertheless, the growth rate on each sugar remained similar. Conclusions C. kronotskyensis possesses a xylose transporter that might take up glucose at an inferior rate even in the absence of xylose. Alternatively, glucose can be taken up in the form of cellobiose, but growth performance is still inferior to growth on xylose. Therefore, we propose that the catabolism of C. kronotskyensis has adapted more strongly to pentose rather than hexose, thereby having obtained a specific survival edge in thermophilic lignocellulosic degradation communities.

Background The members of the genus Caldicellulosiruptor have the potential for future integration into a biorefinery system due to their capacity to generate hydrogen close to the theoretical limit of 4 mol H2/mol hexose, use a wide range of sugars and can grow on numerous lignocellulose hydrolysates. However, members of this genus are unable to survive in high sugar concentrations, limiting their ability to grow on more concentrated hydrolysates, thus impeding their industrial applicability. In this study five members of this genus, C.owensensis, C. kronotskyensis, C.bescii, C.acetigenus and C.kristjanssonii, were developed to tolerate higher sugar concentrations through an adaptive laboratory evolution (ALE) process. The developed mixed population C.owensensis CO80 was further studied and accompanied by the development of a kinetic model based on Monod kinetics to quantitatively compare it with the parental strain. Results Mixed populations of Caldicellulosiruptor tolerant to higher glucose concentrations were obtained with C.owensensis adapted to grow up to 80 g/L glucose; other strains in particular C. kristjanssonii demonstrated a greater restriction to adaptation. The C.owensensis CO80 mixed population was further studied and demonstrated the ability to grow in glucose concentrations up to 80 g/L glucose, but with reduced volumetric hydrogen productivities ($$Q_{{{\text{H}}_{2} }}$$ Q H 2 ) and incomplete sugar conversion at elevated glucose concentrations. In addition, the carbon yield decreased with elevated concentrations of glucose. The ability of the mixed population C.owensensis CO80 to grow in high glucose concentrations was further described with a kinetic growth model, which revealed that the critical sugar concentration of the cells increased fourfold when cultivated at higher concentrations. When co-cultured with the adapted C.saccharolyticus G5 mixed culture at a hydraulic retention time (HRT) of 20 h, C.owensensis constituted only 0.09–1.58% of the population in suspension. Conclusions The adaptation of members of the Caldicellulosiruptor genus to higher sugar concentrations established that the ability to develop improved strains via ALE is species dependent, with C.owensensis adapted to grow on 80 g/L, whereas C.kristjanssonii could only be adapted to 30 g/L glucose. Although C.owensensis CO80 was adapted to a higher sugar concentration, this mixed population demonstrated reduced $$Q_{{{\text{H}}_{2} }}$$ Q H 2 with elevated glucose concentrations. This would indicate that while ALE permits adaptation to elevated sugar concentrations, this approach does not result in improved fermentation performances at these higher sugar concentrations. Moreover, the observation that planktonic mixed culture of CO80 was outcompeted by an adapted C.saccharolyticus, when co-cultivated in continuous mode, indicates that the robustness of CO80 mixed culture should be improved for industrial application.

Bioethanol production processes with Saccharomyces cerevisiae using lignocellulosic biomass as feedstock are challenged by the simultaneous utilization of pentose and hexose sugars from biomass hydrolysates. The pentose uptake into the cell represents a crucial role for the efficiency of the process. The focus of the here presented study was to understand the uptake and conversion of the pentose l-arabinose in S. cerevisiae and reveal its regulation by d-glucose and d-galactose. Gal2p—the most prominent transporter enabling l-arabinose uptake in S. cerevisiae wild-type strains—has an affinity for the transport of l-arabinose, d-glucose, and d-galactose. d-Galactose was reported for being mandatory for inducing GAL2 expression. GAL2 expression is also known to be regulated by d-glucose-mediated carbon catabolite repression, as well as catabolite inactivation. The results of the present study demonstrate that l-arabinose can be used as sole carbon and energy source by the recombinant industrial strain S. cerevisiae DS61180. RT-qPCR and RNA-Seq experiments confirmed that l-arabinose can trigger its own uptake via the induction of GAL2 expression. Expression levels of GAL2 during growth on l-arabinose reached up to 21% of those obtained with d-galactose as sole carbon and energy source. l-Arabinose-induced GAL2 expression was also subject to catabolite repression by d-glucose. Kinetic investigations of substrate uptake, biomass, and product formation during growth on a mixture of d-glucose/l-arabinose revealed impairment of growth and ethanol production from l-arabinose upon d-glucose depletion. The presence of d-glucose is thus preventing the fermentation of l-arabinose in S. cerevisiae DS61180. Comparative transcriptome studies including the wild-type and a precursor strain delivered hints for an increased demand in ATP production and cofactor regeneration during growth of S. cerevisiae DS61180 on l-arabinose. Our results thus emphasize that cofactor and energy metabolism demand attention if the combined conversion of hexose and pentose sugars is intended, for example in biorefineries using lignocellulosics. Electronic supplementary material The online version of this article (10.1186/s13068-018-1231-8) contains supplementary material, which is available to authorized users.

Background The 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. Results Using 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.” Conclusions In 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 material The online version of this article (doi:10.1186/s13068-017-0705-4) contains supplementary material, which is available to authorized users.

Background In mixed sugar fermentations with recombinant Saccharomyces cerevisiae strains able to ferment D-xylose and L-arabinose the pentose sugars are normally only utilized after depletion of D-glucose. This has been attributed to competitive inhibition of pentose uptake by D-glucose as pentose sugars are taken up into yeast cells by individual members of the yeast hexose transporter family. We wanted to investigate whether D-glucose inhibits pentose utilization only by blocking its uptake or also by interfering with its further metabolism. Results To distinguish between inhibitory effects of D-glucose on pentose uptake and pentose catabolism, maltose was used as an alternative carbon source in maltose-pentose co-consumption experiments. Maltose is taken up by a specific maltose transport system and hydrolyzed only intracellularly into two D-glucose molecules. Pentose consumption decreased by about 20 - 30% during the simultaneous utilization of maltose indicating that hexose catabolism can impede pentose utilization. To test whether intracellular D-glucose might impair pentose utilization, hexo-/glucokinase deletion mutants were constructed. Those mutants are known to accumulate intracellular D-glucose when incubated with maltose. However, pentose utilization was not effected in the presence of maltose. Addition of increasing concentrations of D-glucose to the hexo-/glucokinase mutants finally completely blocked D-xylose as well as L-arabinose consumption, indicating a pronounced inhibitory effect of D-glucose on pentose uptake. Nevertheless, constitutive overexpression of pentose-transporting hexose transporters like Hxt7 and Gal2 could improve pentose consumption in the presence of D-glucose. Conclusion Our results confirm that D-glucose impairs the simultaneous utilization of pentoses mainly due to inhibition of pentose uptake. Whereas intracellular D-glucose does not seem to have an inhibitory effect on pentose utilization, further catabolism of D-glucose can also impede pentose utilization. Nevertheless, the results suggest that co-fermentation of pentoses in the presence of D-glucose can significantly be improved by the overexpression of pentose transporters, especially if they are not inhibited by D-glucose.

The heterotrophic and mixotrophic culture of oleaginous microalgae is a promising process to produce biofuel feedstock due to the advantage of fast growth. Various organic carbons have been explored for this application. However, despite being one of the most abundant and economical sugar resources in nature, D-xylose has never been demonstrated as a carbon source for wild-type microalgae. The purpose of the present work was to identify the feasibility of D-xylose utilization by the oleaginous microalga Chlorella sorokiniana. The sugar uptake kinetic analysis was performed with 14C-labeled sugars and the data showed that the D-glucose induced algal cells (the alga was heterotrophically grown on D-glucose and then harvested as D-glucose induced cells) exhibited a remarkably increased D-xylose uptake rate. The maximum D-xylose transport rate was 3.8 nmol min-1 mg-1 dry cell weight (DCW) with K m value of 6.8 mM. D-xylose uptake was suppressed in the presence of D-glucose, D-galactose and D-fructose but not L-arabinose and D-ribose. The uptake of D-xylose activated the related metabolic pathway, and the activities of a NAD(P)H-linked xylose reductase (XR) and a unique NADP+-linked xylitol dehydrogenase (XDH) were detected in C. sorokiniana. Compared with the culture in the dark, the consumption of D-xylose increased 2 fold under light but decreased to the same level with addition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), indicating that extra chemical energy from the light-dependent reaction contributed the catabolism of D-xylose for C. sorokiniana. An inducible D-xylose transportation system and a related metabolic pathway were discovered for microalga for the first time. The transportation of D-xylose across the cell membrane of C. sorokiniana could be realized by an inducible hexose symporter. The uptake of D-xylose subsequently activated the expression of key catalytic enzymes that enabled D-xylose entering central metabolism. Results of this research are useful to better understand the D-xylose metabolic pathway in the microalga C. sorokiniana and provide a target for genetic engineering to improve D-xylose utilization for microalgal lipid production.

Background Pure bacterial strains give better yields when producing H2 than mixed, natural communities. However the main drawback with the pure cultures is the need to perform the fermentations under sterile conditions. Therefore, H2 production using artificial co-cultures, composed of well characterized strains, is one of the directions currently undertaken in the field of biohydrogen research. Results Four pure Clostridium cultures, including C. butyricum CWBI1009, C. pasteurianum DSM525, C. beijerinckii DSM1820 and C. felsineum DSM749, and three different co-cultures composed of (1) C. pasteurianum and C. felsineum, (2) C. butyricum and C. felsineum, (3) C. butyricum and C. pasteurianum, were grown in 20 L batch bioreactors. In the first part of the study a strategy composed of three-culture sequences was developed to determine the optimal pH for H2 production (sequence 1); and the H2-producing potential of each pure strain and co-culture, during glucose (sequence 2) and starch (sequence 3) fermentations at the optimal pH. The best H2 yields were obtained for starch fermentations, and the highest yield of 2.91 mol H2/ mol hexose was reported for C. butyricum. By contrast, the biogas production rates were higher for glucose fermentations and the highest value of 1.5 L biogas/ h was observed for the co-culture (1). In general co-cultures produced H2 at higher rates than the pure Clostridium cultures, without negatively affecting the H2 yields. Interestingly, all the Clostridium strains and co-cultures were shown to utilize lactate (present in a starch-containing medium), and C. beijerinckii was able to re-consume formate producing additional H2. In the second part of the study the co-culture (3) was used to produce H2 during 13 days of glucose fermentation in a sequencing batch reactor (SBR). In addition, the species dynamics, as monitored by qPCR (quantitative real-time PCR), showed a stable coexistence of C. pasteurianum and C. butyricum during this fermentation. Conclusions The four pure Clostridium strains and the artificial co-cultures tested in this study were shown to efficiently produce H2 using glucose and starch as carbon sources. The artificial co-cultures produced H2 at higher rates than the pure strains, while the H2 yields were only slightly affected.