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Background: Crude glycerol (CG) and hemicellulose hydrolysate (HH) are low—value side-products of biodiesel transesterification and pulp—and paper industry or lignocellulosic ethanol production, respectively, which can be converted to microbial lipids by oleaginous yeasts. This study aimed to test the ability of oleaginous yeasts to utilise CG and HH and mixtures of them as carbon source. Results: Eleven out of 27 tested strains of oleaginous yeast species were able to grow in plate tests on CG as sole carbon source. Among them, only one ascomycetous strain, belonging to Lipomyces starkeyi, was identified, the other 10 strains were Rhodotorula spec. When yeasts were cultivated in mixed CG/ HH medium, we observed an activation of glycerol conversion in the Rhodotorula strains, but not in L. starkeyi. Two strains—Rhodotorula toruloides CBS 14 and Rhodotorula glutinis CBS 3044 were further tested in controlled fermentations in bioreactors in different mixtures of CG and HH. The highest measured average biomass and lipid concentration were achieved with R. toruloides in 10% HH medium mixed with 55 g/L CG—19.4 g/L and 10.6 g/L, respectively, with a lipid yield of 0.25 g lipids per consumed g of carbon source. Fatty acid composition was similar to other R. toruloides strains and comparable to that of vegetable oils. Conclusions: There were big strain differences in the ability to convert CG to lipids, as only few of the tested strains were able to grow. Lipid production rates and yields showed that mixing GC and HH have a stimulating effect on lipid accumulation in R. toruloides and R. glutinis resulting in shortened fermentation time to reach maximum lipid concentration, which provides a new perspective on converting these low-value compounds to microbial lipids. [ABSTRACT FROM AUTHOR]

The glycoside hydrolase family 3 (GH3) β‐glucosidases are a structurally diverse family of enzymes. Cel3A from Neurospora crassa (NcCel3A) belongs to a subfamily of key enzymes that are crucial for industrial biomass degradation. β‐Glucosidases hydrolyse the β‐1,4 bond at the nonreducing end of cellodextrins. The hydrolysis of cellobiose is of special importance as its accumulation inhibits other cellulases acting on crystalline cellulose. Here, the crystal structure of the biologically relevant dimeric form of NcCel3A is reported. The structure has been refined to 2.25 Å resolution, with an Rcryst and Rfree of 0.18 and 0.22, respectively. NcCel3A is an extensively N‐glycosylated glycoprotein that shares 46% sequence identity with Hypocrea jecorina Cel3A, the structure of which has recently been published, and 61% sequence identity with the thermophilic β‐glucosidase from Rasamsonia emersonii. NcCel3A is a three‐domain protein with a number of extended loops that deepen the active‐site cleft of the enzyme. These structures characterize this subfamily of GH3 β‐glucosidases and account for the high cellobiose specificity of this subfamily. The glycoside hydrolase family 3 β‐glucosidase Cel3A from Neurospora crassa (NcCel3A) is highly specific for cellobiose. The crystal structure of NcCel3A has been solved to 2.25 Å resolution. The structure is a dimer and exhibits a high degree of N‐glycosylation. [ABSTRACT FROM AUTHOR]

Background: The ascomycete fungus Trichoderma reesei is the predominant source of enzymes for industrial conversion of lignocellulose. Its glycoside hydrolase family 7 cellobiohydrolase (GH7 CBH) TreCel7A constitutes nearly half of the enzyme cocktail by weight and is the major workhorse in the cellulose hydrolysis process. The orthologs from Trichoderma atroviride (TatCel7A) and Trichoderma harzianum (ThaCel7A) show high sequence identity with TreCel7A, ~ 80%, and represent naturally evolved combinations of cellulose-binding tunnel-enclosing loop motifs, which have been suggested to influence intrinsic cellobiohydrolase properties, such as endo-initiation, processivity, and off-rate. Results: The TatCel7A, ThaCel7A, and TreCel7A enzymes were characterized for comparison of function. The catalytic domain of TatCel7A was crystallized, and two structures were determined: without ligand and with thio-cellotriose in the active site. Initial hydrolysis of bacterial cellulose was faster with TatCel7A than either ThaCel7A or TreCel7A. In synergistic saccharification of pretreated corn stover, both TatCel7A and ThaCel7A were more efficient than TreCel7A, although TatCel7A was more sensitive to thermal inactivation. Structural analyses and molecular dynamics (MD) simulations were performed to elucidate important structure/function correlations. Moreover, reverse conservation analysis (RCA) of sequence diversity revealed divergent regions of interest located outside the cellulose-binding tunnel of Trichoderma spp. GH7 CBHs. Conclusions: We hypothesize that the combination of loop motifs is the main determinant for the observed differences in Cel7A activity on cellulosic substrates. Fine-tuning of the loop flexibility appears to be an important evolutionary target in Trichoderma spp., a conclusion supported by the RCA data. Our results indicate that, for industrial use, it would be beneficial to combine loop motifs from TatCel7A with the thermostability features of TreCel7A. Furthermore, one region implicated in thermal unfolding is suggested as a primary target for protein engineering. [ABSTRACT FROM AUTHOR]

This study investigated lipid production from the hemicellulosic fraction of birch wood by the oleaginous yeast Lipomyces starkeyi. Birch wood chips were thermochemically pretreated by hot water extraction, and the liquid phase, containing 45.1 g/l xylose as the major sugar, 13.1 g/l acetic acid and 4.7 g/l furfural, was used for cultivations of L. starkeyi CBS1807. The hydrolysate strongly inhibited yeast growth; the strain could only grow in medium containing 30% hydrolysate at pH 6. At pH 5, growth stopped already upon the addition of about 10% hydrolysate. In fed-batch cultures fed with hydrolysate or a model xylose-acetic acid mixture, co-consumption of xylose and acetic acid was observed, which resulted in a pH increase. This phenomenon was utilized to establish a pH-stat fed-batch cultivation in which, after an initial feeding, hydrolysate or model mixture was connected to the pH-regulation system of the bioreactor. Under these conditions we obtained growth and lipid production in cultures grown on either xylose or glucose during the batch phase. In cultivations fed with model mixture, a maximum lipid content of 60.5% of the cell dry weight (CDW) was obtained; however, not all xylose was consumed. When feeding hydrolysate, growth was promoted and carbon sources were completely consumed, resulting in higher CDW with maximum lipid content of 51.3%. In both cultures the lipid concentration was 8 g/l and a lipid yield of 0.1 g/g carbon source was obtained. Lipid composition was similar in all cultivations, with C18:1 and C16:0 being the most abundant fatty acids. Copyright © 2016 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

The filamentous fungus Hypocrea jecorina (anamorph of Trichoderma reesei) is the predominant source of enzymes for industrial saccharification of lignocellulose biomass. The major enzyme, cellobiohydrolase Cel7A, constitutes nearly half of the total protein in the secretome. The performance of such enzymes is susceptible to inhibition by compounds liberated by physico-chemical pre-treatment if the biomass is kept unwashed. Xylan and xylo-oligosaccharides ( XOS) have been proposed to play a key role in inhibition of cellobiohydrolases of glycoside hydrolase family 7. To elucidate the mechanism behind this inhibition at a molecular level, we used X-ray crystallography to determine structures of H. jecorina Cel7A in complex with XOS. Structures with xylotriose, xylotetraose and xylopentaose revealed a predominant binding mode at the entrance of the substrate-binding tunnel of the enzyme, in which each xylose residue is shifted ~ 2.4 Å towards the catalytic center compared with binding of cello-oligosaccharides. Furthermore, partial occupancy of two consecutive xylose residues at subsites -2 and -1 suggests an alternative binding mode for XOS in the vicinity of the catalytic center. Interestingly, the -1 xylosyl unit exhibits an open aldehyde conformation in one of the structures and a ring-closed pyranoside in another complex. Complementary inhibition studies with p-nitrophenyl lactoside as substrate indicate mixed inhibition rather than pure competitive inhibition. Database The atomic coordinates and structure factors are available in the Protein Data Bank under accession number ( H. jecorina Cel7A E212Q variant, complex with xylotriose), ( H. jecorina Cel7A E217Q variant, complex with xylotriose), ( H. jecorina Cel7A E212Q variant, complex with xylopentaose), ( H. jecorina Cel7A E217Q variant, complex with xylopentaose), (wild-type H. jecorina Cel7A, complex with xylopentaose) and ( H. jecorina Cel7A E217Q variant, complex with xylotetraose). [ABSTRACT FROM AUTHOR]

Plant cell-wall polysaccharides represent a vast source of food in nature. To depolymerize polysaccharides to soluble sugars, many organisms use multifunctional enzyme mixtures consisting of glycoside hydrolases, lytic polysaccharide mono-oxygenases, polysaccharide lyases, and carbohydrate esterases, as well as accessory, redox-active enzymes for lignin depolymerization. Many of these enzymes that degrade lignocellulose are multimodular with carbohydrate-binding modules (CBMs) and catalytic domains connected by flexible, glycosylated linkers. These linkers have long been thought to simply serve as a tether between structured domains or to act in an inchworm-like fashion during catalytic action. To examine linker function, we performed molecular dynamics (MD) simulations of the Trichoderma reesei Family 6 and Family 7 cellobiohydrolases (TrCel6A and TrCel7A, respectively) bound to cellulose. During these simulations, the glycosylated linkers bind directly to cellulose, suggesting a previously unknown role in enzyme action. The prediction from the MD simulations was examined experimentally by measuring the binding affinity of the Cel7A CBM and the natively glycosylated Cel7A CBM-linker. On crystalline cellulose, the glycosylated linker enhances the binding affinity over the CBM alone by an order of magnitude. The MD simulations before and after binding of the linker also suggest that the bound linker may affect enzyme action due to significant damping in the enzyme fluctuations. Together, these results suggest that glycosylated linkers in carbohydrate-active enzymes, which are intrinsically disordered proteins in solution, aid in dynamic binding during the enzymatic deconstruction of plant cell walls. [ABSTRACT FROM AUTHOR]

Background: Salix spp. are high-productivity crops potentially used for lignocellulosic biofuels such as bioethanol. In general, pretreatment is needed to facilitate the enzymatic depolymerization process. Biomass resistance to degradation, i.e., biomass recalcitrance, is a trait which can be assessed by measuring the sugar released after combined pretreatment and enzymatic hydrolysis. We have examined genetic parameters of enzymatic sugar release and other traits related to biorefinery use in a population of 286 natural Salix viminalis clones. Furthermore, we have evaluated phenotypic and genetic correlations between these traits and performed a genomewide association mapping analysis using a set of 19,411 markers. Results: Sugar release (glucose and xylose) after pretreatment and enzymatic saccharification proved highly variable with large genetic and phenotypic variations, and chip heritability estimates (h2) of 0.23–0.29. Lignin syringyl/guaiacyl (S/G) ratio and wood density were the most heritable traits (h2 = 0.42 and 0.59, respectively). Sugar release traits were positively correlated, phenotypically and genetically, with biomass yield and lignin S/G ratio. Association mapping revealed seven marker–trait associations below a suggestive significance threshold, including one marker associated with glucose release. Conclusions: We identified lignin S/G ratio and shoot diameter as heritable traits that could be relatively easily evaluated by breeders, making them suitable proxy traits for developing low-recalcitrance varieties. One marker below the suggestive threshold for marker associations was identified for sugar release, meriting further investigation while also highlighting the difficulties in employing genomewide association mapping for complex traits. [ABSTRACT FROM AUTHOR]

ß-Glucosidases enhance enzymatic biomass conversion by relieving cellobiose inhibition of endoglucanases and cellobiohydrolases. However, the susceptibility of these enzymes to inhibition and transglycosylation at high glucose or cellobiose concentrations severely limits their activity and, consequently, the overall efficiency of enzyme mixtures. We determined the impact of these two processes on the hydrolytic activity of the industrially relevant family 3 ß-glucosidases from Hypocrea jecorina, HjCel3A and HjCel3B, and investigated the underlying molecular mechanisms through kinetic studies, binding free energy calculations, and molecular dynamics (MD) simulations. HjCel3B had a 7-fold higher specificity for cellobiose than HjCel3A but greater tendency for glucose inhibition. Energy decomposition analysis indicated that cellobiose has relatively weak electrostatic interactions with binding site residues, allowing it to be easily displaced by glucose and free to inhibit other hydrolytic enzymes. HjCel3A is, thus, preferable as an industrial ß-glucosidase despite its lower activity caused by transglycosylation. This competing pathway to hydrolysis arises from binding of glucose or cellobiose at the product site after formation of the glycosyl-enzyme intermediate. MD simulations revealed that binding is facilitated by hydrophobic interactions with Trp-37, Phe-260, and Tyr-443. Targeting these aromatic residues for mutation to reduce substrate affinity at the product site would therefore potentially mitigate transglycosidic activity. Engineering improved variants of HjCel3A and other structurally similar ß-glucosidases would have a significant economic effect on enzymatic biomass conversion in terms of yield and production cost as the process can be consequently conducted at higher substrate loadings. [ABSTRACT FROM AUTHOR]

Abstract: Using standard laboratory equipment, thermochemically pretreated oat straw was enzymatically saccharified and fermented to ethanol, and after removal of ethanol the remaining material was subjected to biogas digestion. A detailed mass balance calculation shows that, for steam explosion pretreatment, this combined ethanol fermentation and biogas digestion converts 85–87% of the higher heating value (HHV) of holocellulose (cellulose and hemicellulose) in the oat straw into biofuel energy. The energy (HHV) yield of the produced ethanol and methane was 9.5–9.8MJ/(kg dry oat straw), which is 28–34% higher than direct biogas digestion that yielded 7.3–7.4MJ/(kg dry oat straw). The rate of biogas formation from the fermentation residues was also higher than from the corresponding pretreated but unfermented oat straw, indicating that the biogas digestion could be terminated after only 24days. This suggests that the ethanol process acts as an additional pretreatment for the biogas process. [Copyright &y& Elsevier]