The first crystal structure of a family 45 glycoside hydrolase from a brown-rot fungus, Gloeophyllum trabeum GtCel45A.

Here we describe the first crystal structure of a beta-1,4-endoglucanase from a brown-rot fungus, Gloeophyllum trabeum GtCel45A, which belongs to subfamily C of glycoside hydrolase family 45 (GH45). GtCel45A is ~ 18 kDa in size and the crystal structure contains 179 amino acids. The structure is refined at 1.30 Å resolution and Rfree 0.18. The enzyme consists of a single catalytic module folded into a six-stranded double-psi beta-barrel domain surrounded by long loops. GtCel45A is very similar in sequence (82% identity) and structure to PcCel45A from the white-rot fungus Phanerochaete chrysosporium. Surprisingly though, initial hydrolysis of barley beta-glucan was almost twice as fast in GtCel45A as compared to PcCel45A.

SPIRO - the automated Petri plate imaging platform designed by biologists, for biologists.

Phenotyping of model organisms grown on Petri plates is often carried out manually, despite the procedures being time-consuming and laborious. The main reason for this is the limited availability of automated phenotyping facilities, whereas constructing a custom automated solution can be a daunting task for biologists. Here, we describe SPIRO, the Smart Plate Imaging Robot, an automated platform that acquires time-lapse photographs of up to four vertically oriented Petri plates in a single experiment, corresponding to 192 seedlings for a typical root growth assay and up to 2500 seeds for a germination assay. SPIRO is catered specifically to biologists' needs, requiring no engineering or programming expertise for assembly and operation. Its small footprint is optimized for standard incubators, the inbuilt green LED enables imaging under dark conditions, and remote control provides access to the data without interfering with sample growth. SPIRO's excellent image quality is suitable for automated image processing, which we demonstrate on the example of seed germination and root growth assays. Furthermore, the robot can be easily customized for specific uses, as all information about SPIRO is released under open-source licenses. Importantly, uninterrupted imaging allows considerably more precise assessment of seed germination parameters and root growth rates compared with manual assays. Moreover, SPIRO enables previously technically challenging assays such as phenotyping in the dark. We illustrate the benefits of SPIRO in proof-of-concept experiments which yielded a novel insight on the interplay between autophagy, nitrogen sensing, and photoblastic response.

The crystal structure of RsSymEG1 reveals a unique form of smaller GH7 endoglucanases alongside GH7 cellobiohydrolases in protist symbionts of termites.

Glycoside hydrolase family 7 (GH7) cellulases are key enzymes responsible for carbon cycling on earth through their role in cellulose degradation and constitute highly important industrial enzymes as well. Although these enzymes are found in a wide variety of evolutionarily distant organisms across eukaryotes, they exhibit remarkably conserved features within two groups: exo-acting cellobiohydrolases and endoglucanases. However, recently reports have emerged of a separate clade of GH7 endoglucanases from protist symbionts of termites that are 60-80 amino acids shorter. In this work, we describe the first crystal structure of a short GH7 endoglucanase, RsSymEG1, from a symbiont of the lower termite Reticulitermes speratus. A more open flat surface and shorter loops around the non-reducing end of the cellulose-binding cleft indicate enhanced access to cellulose chains on the surface of cellulose microfibrils. Additionally, when comparing activities on polysaccharides to a typical fungal GH7 endoglucanase (Trichoderma longibrachiatum Cel7B), RsSymEG1 showed significantly faster initial hydrolytic activity. We also examine the prevalence and diversity of GH7 enzymes that the symbionts provide to the termite host, compare overall structures and substrate binding between cellobiohydrolase and long and short endoglucanase, and highlight the presence of similar short GH7s in other organisms.

Energy performance of compressed biomethane gas production from co-digestion of Salix and dairy manure: factoring differences between Salix varieties.

Biogas from anaerobic digestion is a versatile energy carrier that can be upgraded to compressed biomethane gas (CBG) as a renewable and sustainable alternative to natural gas. Organic residues and energy crops are predicted to be major sources of bioenergy production in the future. Pre-treatment can reduce the recalcitrance of lignocellulosic energy crops such as Salix to anaerobic digestion, making it a potential biogas feedstock. This lignocellulosic material can be co-digested with animal manure, which has the complementary effect of increasing volumetric biogas yield. Salix varieties exhibit variations in yield, composition and biomethane potential values, which can have a significant effect on the overall biogas production system. This study assessed the impact of Salix varietal differences on the overall mass and energy balance of a co-digestion system using steam pre-treated Salix biomass and dairy manure (DaM) to produce CBG as the final product. Six commercial Salix varieties cultivated under unfertilised and fertilised conditions were compared. Energy and mass flows along this total process chain, comprising Salix cultivation, steam pre-treatment, biogas production and biogas upgrading to CBG, were evaluated. Two scenarios were considered: a base scenario without heat recovery and a scenario with heat recovery. The results showed that Salix variety had a significant effect on energy output-input ratio (R), with R values in the base scenario of 1.57-1.88 and in the heat recovery scenario of 2.36-2.94. In both scenarios, unfertilised var. Tordis was the best energy performer, while the fertilised var. Jorr was the worst. Based on this energy performance, Salix could be a feasible feedstock for co-digestion with DaM, although its R value was at the lower end of the range reported previously for energy crops.

Sustainable Delicacy: Variation in Quality and Sensory Aspects in Wild Boar (Sus scrofa) Meat and Comparison to Pork Meat-A Case Study.

The aim of this study was to evaluate quality and sensory variation in wild boar meat in comparison to pork. Meat quality in wild boar is expected to vary more compared to pork due to different feeding environment, age and gender. In order to be able to promote wild boar meat as a sustainable high-quality product, there is a need to evaluate the variation in meat quality attributes, including technological, compositional and sensory/texture aspects. We evaluated carcass characteristics, pH, colour, lipid profile and sensory aspects of wild boar meat of different age and gender and compared them with pork. Wild boars had lower carcass weight (p = <0.0001) and higher ultimate pH (p = 0.0063) compared to domestic pigs. Intramuscular fat content had a tendency to be higher in wild boar meat (p = 0.1010), as well as the proportion of nutritional valuable n-3 FA (p = 0.0029). The colour of pork was more pink (p = 0.0276) and pale (p = <0.0001) compared to meat from wild boar. Meat from wild boar gilts received the highest sensory scores. Based on these findings, we suggest that meat from younger animals could be sold in different cuts directly while meat from older animals might be more suitable for the production of sausage.

Comparison of glycoside hydrolase family 3 ß-xylosidases from basidiomycetes and ascomycetes reveals evolutionarily distinct xylan degradation systems.

Xylan is the most common hemicellulose in plant cell walls, though the structure of xylan polymers differs between plant species. Here, to gain a better understanding of fungal xylan degradation systems, which can enhance enzymatic saccharification of plant cell walls in industrial processes, we conducted a comparative study of two glycoside hydrolase family 3 (GH3) ß-xylosidases (Bxls), one from the basidiomycete Phanerochaete chrysosporium (PcBxl3), and the other from the ascomycete Trichoderma reesei (TrXyl3A). A comparison of the crystal structures of the two enzymes, both with saccharide bound at the catalytic center, provided insight into the basis of substrate binding at each subsite. PcBxl3 has a substrate-binding pocket at subsite -1, while TrXyl3A has an extra loop that contains additional binding subsites. Furthermore, kinetic experiments revealed that PcBxl3 degraded xylooligosaccharides faster than TrXyl3A, while the KM values of TrXyl3A were lower than those of PcBxl3. The relationship between substrate specificity and degree of polymerization of substrates suggested that PcBxl3 preferentially degrades xylobiose (X2), while TrXyl3A degrades longer xylooligosaccharides. Moreover, docking simulation supported the existence of extended positive subsites of TrXyl3A in the extra loop located at the N-terminus of the protein. Finally, phylogenetic analysis suggests that wood-decaying basidiomycetes use Bxls such as PcBxl3 that act efficiently on xylan structures from woody plants, whereas molds use instead Bxls that efficiently degrade xylan from grass. Our results provide added insights into fungal efficient xylan degradation systems.

Glucomannan and beta-glucan degradation by Mytilus edulis Cel45A: Crystal structure and activity comparison with GH45 subfamily A, B and C.

The enzymatic hydrolysis of barley beta-glucan, konjac glucomannan and carboxymethyl cellulose by a ß-1,4-D-endoglucanase MeCel45A from blue mussel, Mytilus edulis, which belongs to subfamily B of glycoside hydrolase family 45 (GH45), was compared with GH45 members of subfamilies A (Humicola insolens HiCel45A), B (Trichoderma reesei TrCel45A) and C (Phanerochaete chrysosporium PcCel45A). Furthermore, the crystal structure of MeCel45A is reported. Initial rates and hydrolysis yields were determined by reducing sugar assays and product formation was characterized using NMR spectroscopy. The subfamily B and C enzymes exhibited mannanase activity, whereas the subfamily A member was uniquely able to produce monomeric glucose. All enzymes were confirmed to be inverting glycoside hydrolases. MeCel45A appears to be cold adapted by evolution, as it maintained 70% activity on cellohexaose at 4 °C relative to 30 °C, compared to 35% for TrCel45A. Both enzymes produced cellobiose and cellotetraose from cellohexaose, but TrCel45A additionally produced cellotriose.

Machine learning reveals sequence-function relationships in family 7 glycoside hydrolases.

Family 7 glycoside hydrolases (GH7) are among the principal enzymes for cellulose degradation in nature and industrially. These enzymes are often bimodular, including a catalytic domain and carbohydrate-binding module (CBM) attached via a flexible linker, and exhibit an active site that binds cello-oligomers of up to ten glucosyl moieties. GH7 cellulases consist of two major subtypes: cellobiohydrolases (CBH) and endoglucanases (EG). Despite the critical importance of GH7 enzymes, there remain gaps in our understanding of how GH7 sequence and structure relate to function. Here, we employed machine learning to gain data-driven insights into relationships between sequence, structure, and function across the GH7 family. Machine-learning models, trained only on the number of residues in the active-site loops as features, were able to discriminate GH7 CBHs and EGs with up to 99% accuracy, demonstrating that the lengths of loops A4, B2, B3, and B4 strongly correlate with functional subtype across the GH7 family. Classification rules were derived such that specific residues at 42 different sequence positions each predicted the functional subtype with accuracies surpassing 87%. A random forest model trained on residues at 19 positions in the catalytic domain predicted the presence of a CBM with 89.5% accuracy. Our machine learning results recapitulate, as top-performing features, a substantial number of the sequence positions determined by previous experimental studies to play vital roles in GH7 activity. We surmise that the yet-to-be-explored sequence positions among the top-performing features also contribute to GH7 functional variation and may be exploited to understand and manipulate function.

Oleaginous yeasts respond differently to carbon sources present in lignocellulose hydrolysate.

BACKGROUND: Microbial oils, generated from lignocellulosic material, have great potential as renewable and sustainable alternatives to fossil-based fuels and chemicals. By unravelling the diversity of lipid accumulation physiology in different oleaginous yeasts grown on the various carbon sources present in lignocellulose hydrolysate (LH), new targets for optimisation of lipid accumulation can be identified. Monitoring lipid formation over time is essential for understanding lipid accumulation physiology. This study investigated lipid accumulation in a variety of oleaginous ascomycetous and basidiomycetous strains grown in glucose and xylose and followed lipid formation kinetics of selected strains in wheat straw hydrolysate (WSH). RESULTS: Twenty-nine oleaginous yeast strains were tested for their ability to utilise glucose and xylose, the main sugars present in WSH. Evaluation of sugar consumption and lipid accumulation revealed marked differences in xylose utilisation capacity between the yeast strains, even between those belonging to the same species. Five different promising strains, belonging to the species Lipomyces starkeyi, Rhodotorula glutinis, Rhodotorula babjevae and Rhodotorula toruloides, were grown on undiluted wheat straw hydrolysate and lipid accumulation was followed over time, using Fourier transform-infrared (FTIR) spectroscopy. All five strains were able to grow on undiluted WSH and to accumulate lipids, but to different extents and with different productivities. R. babjevae DVBPG 8058 was the best-performing strain, accumulating 64.8% of cell dry weight (CDW) as lipids. It reached a culture density of 28 g/L CDW in batch cultivation, resulting in a lipid content of 18.1 g/L and yield of 0.24 g lipids per g carbon source. This strain formed lipids from the major carbon sources in hydrolysate, glucose, acetate and xylose. R. glutinis CBS 2367 also consumed these carbon sources, but when assimilating xylose it consumed intracellular lipids simultaneously. Rhodotorula strains contained a higher proportion of polyunsaturated fatty acids than the two tested Lipomyces starkeyi strains. CONCLUSIONS: There is considerable metabolic diversity among oleaginous yeasts, even between closely related species and strains, especially when converting xylose to biomass and lipids. Monitoring the kinetics of lipid accumulation and identifying the molecular basis of this diversity are keys to selecting suitable strains for high lipid production from lignocellulose.

Microbial lipid production from crude glycerol and hemicellulosic hydrolysate with oleaginous yeasts.

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.

Enhanced detection of ATTR amyloid using a nanofibril-based assay.

More than 30 proteins and peptides have been found to form amyloid fibrils in human diseases. Fibrils formed by transthyretin (TTR) are associated with ATTR amyloidosis, affecting many vital organs, including the heart and peripheral nervous system. Congo red staining is the gold standard method for detection of amyloid deposits in tissue. However, Congo red staining and amyloid typing methods such as immunofluorescence labelling are limited to relatively large deposits. Detection of small ATTR deposits present at an early stage of the disease could enable timely treatment and prevent severe tissue damage. In this study, we developed an enhanced ATTR amyloid detection method that uses functionalised protein nanofibrils. Using this method, we achieved sensitive detection of monomeric TTR in a microplate immunoassay and immunofluorescence labelling of ex vivo tissue from two patients containing ATTR aggregates. The system's utility was confirmed on sections from a patient with AA amyloidosis and liver sections from inflamed mouse. These results suggest that the detection system constitutes important new technology for highly sensitive detection of microscopic amounts of ATTR amyloid deposited in tissue.

Assembly and Analysis of the Genome Sequence of the Yeast Brettanomyces naardenensis CBS 7540.

Brettanomyces naardenensis is a spoilage yeast with potential for biotechnological applications for production of innovative beverages with low alcohol content and high attenuation degree. Here, we present the first annotated genome of B. naardenensis CBS 7540. The genome of B. naardenensis CBS 7540 was assembled into 76 contigs, totaling 11,283,072 nucleotides. In total, 5168 protein-coding sequences were annotated. The study provides functional genome annotation, phylogenetic analysis, and discusses genetic determinants behind notable stress tolerance and biotechnological potential of B. naardenensis.

The dissociation mechanism of processive cellulases.

Cellulase enzymes deconstruct recalcitrant cellulose into soluble sugars, making them a biocatalyst of biotechnological interest for use in the nascent lignocellulosic bioeconomy. Cellobiohydrolases (CBHs) are cellulases capable of liberating many sugar molecules in a processive manner without dissociating from the substrate. Within the complete processive cycle of CBHs, dissociation from the cellulose substrate is rate limiting, but the molecular mechanism of this step is unknown. Here, we present a direct comparison of potential molecular mechanisms for dissociation via Hamiltonian replica exchange molecular dynamics of the model fungal CBH, Trichoderma reesei Cel7A. Computational rate estimates indicate that stepwise cellulose dethreading from the binding tunnel is 4 orders of magnitude faster than a clamshell mechanism, in which the substrate-enclosing loops open and release the substrate without reversing. We also present the crystal structure of a disulfide variant that covalently links substrate-enclosing loops on either side of the substrate-binding tunnel, which constitutes a CBH that can only dissociate via stepwise dethreading. Biochemical measurements indicate that this variant has a dissociation rate constant essentially equivalent to the wild type, implying that dethreading is likely the predominant mechanism for dissociation.

Genome-scale model of Rhodotorula toruloides metabolism.

The basidiomycete red yeast Rhodotorula toruloides is a promising platform organism for production of biooils. We present rhto-GEM, the first genome-scale model (GEM) of R. toruloides metabolism, that was largely reconstructed using RAVEN toolbox. The model includes 852 genes, 2,731 reactions, and 2,277 metabolites, while lipid metabolism is described using the SLIMEr formalism allowing direct integration of lipid class and acyl chain experimental distribution data. The simulation results confirmed that the R. toruloides model provides valid growth predictions on glucose, xylose, and glycerol, while prediction of genetic engineering targets to increase production of linolenic acid, triacylglycerols, and carotenoids identified genes-some of which have previously been engineered to successfully increase production. This renders rtho-GEM valuable for future studies to improve the production of other oleochemicals of industrial relevance including value-added fatty acids and carotenoids, in addition to facilitate system-wide omics-data analysis in R. toruloides. Expanding the portfolio of GEMs for lipid-accumulating fungi contributes to both understanding of metabolic mechanisms of the oleaginous phenotype but also uncover particularities of the lipid production machinery in R. toruloides.

Manganese and iron deficiency in Southern Ocean Phaeocystis antarctica populations revealed through taxon-specific protein indicators.

Iron and light are recognized as limiting factors controlling Southern Ocean phytoplankton growth. Recent field-based evidence suggests, however, that manganese availability may also play a role. Here we examine the influence of iron and manganese on protein expression and physiology in Phaeocystis antarctica, a key Antarctic primary producer. We provide taxon-specific proteomic evidence to show that in-situ Southern Ocean Phaeocystis populations regularly experience stress due to combined low manganese and iron availability. In culture, combined low iron and manganese induce large-scale changes in the Phaeocystis proteome and result in reorganization of the photosynthetic apparatus. Natural Phaeocystis populations produce protein signatures indicating late-season manganese and iron stress, consistent with concurrently observed stimulation of chlorophyll production upon additions of manganese or iron. These results implicate manganese as an important driver of Southern Ocean productivity and demonstrate the utility of peptide mass spectrometry for identifying drivers of incomplete macronutrient consumption.

Coupled chemistry kinetics demonstrate the utility of functionalized Sup35 amyloid nanofibrils in biocatalytic cascades.

Concerns over the environment are a central driver for designing cell-free enzymatic cascade reactions that synthesize non-petrol-based commodity compounds. An often-suggested strategy that would demonstrate the economic competitiveness of this technology is recycling of valuable enzymes through their immobilization. For this purpose, amyloid nanofibrils are an ideal scaffold to realize chemistry-free covalent enzyme immobilization on a material that offers a large surface area. However, in most instances, only single enzyme-functionalized amyloid fibrils have so far been studied. To embark on the next stage, here we displayed xylanase A, ß-xylosidase, and an aldose sugar dehydrogenase on Sup35(1-61) nanofibrils to convert beechwood xylan to xylonolactone. We characterized this enzymatic cascade by measuring the time-dependent accumulation of xylose, xylooligomers, and xylonolactone. Furthermore, we studied the effects of relative enzyme concentrations, pH, temperature, and agitation on product formation. Our investigations revealed that a modular cascade with a mixture of xylanase and ß-xylosidase, followed by product removal and separate oxidation of xylose with the aldose sugar dehydrogenase, is more productive than an enzyme mix containing all of these enzymes together. Moreover, we found that the nanofibril-coupled enzymes do not lose activity compared with their native state. These findings provide proof of concept of the feasibility of functionalized Sup35(1-61) fibrils as a molecular scaffold for biocatalytic cascades consisting of reusable enzymes that can be used in biotechnology.

Comparison of three seemingly similar lytic polysaccharide monooxygenases from Neurospora crassa suggests different roles in plant biomass degradation.

Many fungi produce multiple lytic polysaccharide monooxygenases (LPMOs) with seemingly similar functions, but the biological reason for this multiplicity remains unknown. To address this question, here we carried out comparative structural and functional characterizations of three cellulose-active C4-oxidizing family AA9 LPMOs from the fungus Neurospora crassa, NcLPMO9A (NCU02240), NcLPMO9C (NCU02916), and NcLPMO9D (NCU01050). We solved the three-dimensional structure of copper-bound NcLPMO9A at 1.6-Å resolution and found that NcLPMO9A and NcLPMO9C, containing a CBM1 carbohydrate-binding module, bind cellulose more strongly and were less susceptible to inactivation than NcLPMO9D, which lacks a CBM. All three LPMOs were active on tamarind xyloglucan and konjac glucomannan, generating similar products but clearly differing in activity levels. Importantly, in some cases, the addition of phosphoric acid-swollen cellulose (PASC) had a major effect on activity: NcLPMO9A was active on xyloglucan only in the presence of PASC, and PASC enhanced NcLPMO9D activity on glucomannan. Interestingly, the three enzymes also exhibited large differences in their interactions with enzymatic electron donors, which could reflect that they are optimized to act with different reducing partners. All three enzymes efficiently used H2O2 as a cosubstrate, yielding product profiles identical to those obtained in O2-driven reactions with PASC, xyloglucan, or glucomannan. Our results indicate that seemingly similar LPMOs act preferentially on different types of copolymeric substructures in the plant cell wall, possibly because these LPMOs are functionally adapted to distinct niches differing in the types of available reductants.

FT-NIR: a tool for rapid intracellular lipid quantification in oleaginous yeasts.

BACKGROUND: Lipid extraction for quantification of fat content in oleaginous yeasts often requires strong acids and harmful organic solvents; it is laborious and time-consuming. Therefore, in most cases just endpoint measurements of lipid accumulation are performed and kinetics of intracellular lipid accumulation is difficult to follow. To address this, we created a prediction model using Fourier-transform near-infrared (FT-NIR) spectroscopy. This method allows to measure lipid content in yeast. METHODS: The FT-NIR calibration sets were constructed from spectra of freeze-dried cells of the oleaginous yeasts Rhodotorula toruloides CBS 14, Lipomyces starkeyi CBS 1807 and Yarrowia lipolytica CBS 6114. The yeast cells were obtained from different cultivation conditions. Freeze-dried cell pellets were scanned using FT-NIR in the Multi Purpose Analyser (MPA) from Bruker. The obtained spectra were assigned corresponding to total fat content, obtained from lipid extraction using a modified Folch method. Quantification models using partial least squares (PLS) regression were built, and the calibration sets were validated on independently cultivated samples. The R. toruloides model was additionally tested on Rhodotorula babjevae DBVPG 8058 and Rhodotorula glutinis CBS 2387. RESULTS: The R 2 of the FT-NIR model for R. toruloides was 98%, and the root mean square error of cross-validation (RMSECV) was 1.53. The model was validated using a separate set of R. toruloides samples with a root mean square error of prediction (RMSEP) of 3.21. The R 2 of the Lipomyces model was 96%, with RMSECV 2.4 and RMSEP 3.8. The R 2 of the mixed model, including all tested yeast strains, was 90.5%, with RMSECV 2.76 and RMSEP 3.22, respectively. The models were verified by predicting the total fat content in newly cultivated and freeze-dried samples. Additionally, the kinetics of lipid accumulation of a culture were followed and compared with standard lipid extraction methods. CONCLUSIONS: Using FT-NIR spectroscopy, we have developed a faster, less laborious and non-destructive quantification of yeast intracellular lipid content compared to methods using lipid extraction.

Genetic variation of biomass recalcitrance in a natural Salix viminalis (L.) population.

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 (h 2) of 0.23-0.29. Lignin syringyl/guaiacyl (S/G) ratio and wood density were the most heritable traits (h 2 = 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.

Proteome analysis of xylose metabolism in Rhodotorula toruloides during lipid production.

BACKGROUND: Rhodotorula toruloides is a promising platform organism for production of lipids from lignocellulosic substrates. Little is known about the metabolic aspects of lipid production from the lignocellolosic sugar xylose by oleaginous yeasts in general and R. toruloides in particular. This study presents the first proteome analysis of the metabolism of R. toruloides during conversion of xylose to lipids. RESULTS: Rhodotorula toruloides cultivated on either glucose or xylose was subjected to comparative analysis of its growth dynamics, lipid composition, fatty acid profiles and proteome. The maximum growth and sugar uptake rate of glucose-grown R. toruloides cells were almost twice that of xylose-grown cells. Cultivation on xylose medium resulted in a lower final biomass yield although final cellular lipid content was similar between glucose- and xylose-grown cells. Analysis of lipid classes revealed the presence of monoacylglycerol in the early exponential growth phase as well as a high proportion of free fatty acids. Carbon source-specific changes in lipid profiles were only observed at early exponential growth phase, where C18 fatty acids were more saturated in xylose-grown cells. Proteins involved in sugar transport, initial steps of xylose assimilation and NADPH regeneration were among the proteins whose levels increased the most in xylose-grown cells across all time points. The levels of enzymes involved in the mevalonate pathway, phospholipid biosynthesis and amino acids biosynthesis differed in response to carbon source. In addition, xylose-grown cells contained higher levels of enzymes involved in peroxisomal beta-oxidation and oxidative stress response compared to cells cultivated on glucose. CONCLUSIONS: The results obtained in the present study suggest that sugar import is the limiting step during xylose conversion by R. toruloides into lipids. NADPH appeared to be regenerated primarily through pentose phosphate pathway although it may also involve malic enzyme as well as alcohol and aldehyde dehydrogenases. Increases in enzyme levels of both fatty acid biosynthesis and beta-oxidation in xylose-grown cells was predicted to result in a futile cycle. The results presented here are valuable for the development of lipid production processes employing R. toruloides on xylose-containing substrates.