Quantitative comparison of the biomass-degrading enzyme repertoires of five filamentous fungi.

The efficiency of microorganisms to degrade lignified plants is of great importance in the Earth's carbon cycle, but also in industrial biorefinery processes, such as for biofuel production. Here, we present a large-scale proteomics approach to investigate and compare the enzymatic response of five filamentous fungi when grown on five very different substrates: grass (sugarcane bagasse), hardwood (birch), softwood (spruce), cellulose and glucose. The five fungi included the ascomycetes Aspergillus terreus, Trichoderma reesei, Myceliophthora thermophila, Neurospora crassa and the white-rot basidiomycete Phanerochaete chrysosporium, all expressing a diverse repertoire of enzymes. In this study, we present comparable quantitative protein abundance values across five species and five diverse substrates. The results allow for direct comparison of fungal adaptation to the different substrates, give indications as to the substrate specificity of individual carbohydrate-active enzymes (CAZymes), and reveal proteins of unknown function that are co-expressed with CAZymes. Based on the results, we present a quantitative comparison of 34 lytic polysaccharide monooxygenases (LPMOs), which are crucial enzymes in biomass deconstruction.

Enzymatic degradation of sulfite-pulped softwoods and the role of LPMOs.

BACKGROUND: Recent advances in the development of enzyme cocktails for degradation of lignocellulosic biomass, especially the discovery of lytic polysaccharide monooxygenases (LPMOs), have opened new perspectives for process design and optimization. Softwood biomass is an abundant resource in many parts of the world, including Scandinavia, but efficient pretreatment and subsequent enzymatic hydrolysis of softwoods are challenging. Sulfite pulping-based pretreatments, such as in the BALI™ process, yield substrates that are relatively easy to degrade. We have assessed how process conditions affect the efficiency of modern cellulase preparations in processing of such substrates. RESULTS: We show that efficient degradation of sulfite-pulped softwoods with modern, LPMO-containing cellulase preparations requires the use of conditions that promote LPMO activity, notably the presence of molecular oxygen and sufficient reducing power. Under LPMO activity-promoting conditions, glucan conversion after 48-h incubation with Cellic® CTec3 reached 73.7 and 84.3% for Norway spruce and loblolly pine, respectively, at an enzyme loading of 8 mg/g of glucan. The presence of free sulfite ions had a negative effect on hydrolysis efficiency. Lignosulfonates, produced from lignin during sulfite pretreatment, showed a potential to activate LPMOs. Spiking of Celluclast®, a cellulase cocktail with low LPMO activity, with monocomponent cellulases or an LPMO showed that the addition of the LPMO was clearly more beneficial than the addition of any classical cellulase. Addition of the LPMO in reactions with spruce increased the saccharification yield from approximately 60% to the levels obtained with Cellic® CTec3. CONCLUSIONS: In this study, we have demonstrated the importance of LPMOs for efficient enzymatic degradation of sulfite-pulped softwood. We have also shown that to exploit the full potential of LPMO-rich cellulase preparations, conditions promoting LPMO activity, in particular the presence of oxygen and reducing equivalents are necessary, as is removal of residual sulfite from the pretreatment step. The use of lignosulfonates as reductants may reduce the costs related to the addition of small molecule reductants in sulfite pretreatment-based biorefineries.

Development of minimal enzyme cocktails for hydrolysis of sulfite-pulped lignocellulosic biomass.

Despite recent progress, saccharification of lignocellulosic biomass is still a major cost driver in biorefining. In this study, we present the development of minimal enzyme cocktails for hydrolysis of Norway spruce and sugarcane bagasse, which were pretreated using the so-called BALI™ process, which is based on sulfite pulping technology. Minimal enzyme cocktails were composed using several glycoside hydrolases purified from the industrially relevant filamentous fungus Trichoderma reesei and a purified commercial ß-glucosidase from Aspergillus niger. The contribution of in-house expressed lytic polysaccharide monooxygenases (LPMOs) was also tested, since oxidative cleavage of cellulose by such LPMOs is known to be beneficial for conversion efficiency. We show that the optimized cocktails permit efficient saccharification at reasonable enzyme loadings and that the effect of the LPMOs is substrate-dependent. Using a cocktail comprising only four enzymes, glucan conversion for Norway spruce reached >80% at enzyme loadings of 8mg/g glucan, whereas almost 100% conversion was achieved at 16mg/g.

Proteomic investigation of the secretome of Cellvibrio japonicus during growth on chitin.

Studies of the secretomes of microbes grown on insoluble substrates are important for the discovery of novel proteins involved in biomass conversion. However, data in literature and this study indicate that secretome samples tend to be contaminated with cytoplasmic proteins. We have examined the secretome of the Gram-negative soil bacterium Cellvibrio japonicus using a simple plate-based culturing technique that yields samples with high fractions (60-75%) of proteins that are predicted to be secreted. By combining this approach with label-free quantification using the MaxLFQ algorithm, we have mapped and quantified proteins secreted by C. japonicus during growth on α- and ß-chitin. Hierarchical clustering of the detected protein quantities revealed groups of up-regulated proteins that include all five putative C. japonicus chitinases as well as a chitin-specific lytic polysaccharide monooxygenase (CjLPMO10A). A small set of secreted proteins were co-regulated with known chitin-specific enzymes, including several with unknown catalytic functions. These proteins provide interesting targets for further studies aimed at unraveling the enzymatic machineries used by C. japonicus for recalcitrant polysaccharide degradation. Studies of chitin degradation indicated that C. japonicus indeed produces an efficient chitinolytic enzyme cocktail. All MS data have been deposited in the ProteomeXchange with the dataset identifier PXD002843 (http://proteomecentral.proteomexchange.org/dataset/PXD002843).

A novel proteomics sample preparation method for secretome analysis of Hypocrea jecorina growing on insoluble substrates.

Analysis of the secretomes of filamentous fungi growing on insoluble lignocellulosic substrates is of major current interest because of the industrial potential of secreted fungal enzymes. Importantly, such studies can help identifying key enzymes from a large arsenal of bioinformatically detected candidates in fungal genomes. We describe a simple, plate-based method to analyze the secretome of Hypocrea jecorina growing on insoluble substrates that allows harsh sample preparation methods promoting desorption, and subsequent identification, of substrate-bound proteins, while minimizing contamination with non-secreted proteins from leaking or lysed cells. The validity of the method was demonstrated by comparative secretome analysis of wild-type H.jecorina strain QM6a growing on bagasse, birch wood, spruce wood or pure cellulose, using label-fee quantification. The proteomic data thus obtained were consistent with existing data from transcriptomics and proteomics studies and revealed clear differences in the responses to complex lignocellulosic substrates and the response to pure cellulose. This easy method is likely to be generally applicable to filamentous fungi and to other microorganisms growing on insoluble substrates.

Expression of endoglucanases in Pichia pastoris under control of the GAP promoter.

BACKGROUND: Plant-derived biomass is a potential alternative to fossil feedstocks for a greener economy. Enzymatic saccharification of biomass has been studied extensively and endoglucanases have been found to be a prerequisite for quick initial liquefaction of biomass under industrial conditions. Pichia pastoris, widely used for heterologous protein expression, can be utilized for fungal endoglucanase production. The recently marketed PichiaPink™ expression system allows for rapid clone selection, and employs the methanol inducible AOX1 promoter to ensure high protein expression levels. However, methanol is toxic and poses a fire hazard, issues which become more significant at an industrial scale. It is possible to eliminate these risks and still maintain high productivity by switching to the constitutive GAP promoter. RESULTS: In the present study, a plasmid carrying the constitutive GAP promoter was created for PichiaPink™. We then studied expression of two endoglucanases, AfCel12A from Aspergillus fumigatus and TaCel5A from Thermoascus aurantiacus, regulated by either the AOX1 promoter or the GAP promoter. Initial experiments in tubes and small bioreactors showed that the levels of AfCel12A obtained with the constitutive promoter were similar or higher, compared to the AOX1 promoter, whereas the levels of TaCel5A were somewhat lower. After optimization of cultivation conditions using a 15-l bioreactor, the recombinant P. pastoris strains utilizing the GAP promoter produced ca. 3-5 g/l of total secreted protein, with CMCase activity equivalent to 1200 nkat/ml AfCel12A and 170 nkat/ml TaCel5A. CONCLUSIONS: We present a strategy for constitutive recombinant protein expression in the novel PichiaPink™ system. Both AfCel12A and TaCel5A were successfully expressed constitutively in P. pastoris under the GAP promoter. Reasonable protein levels were reached after optimizing cultivation conditions.

The noncommensal bacterium Methylococcus capsulatus (Bath) ameliorates dextran sulfate (Sodium Salt)-Induced Ulcerative Colitis by influencing mechanisms essential for maintenance of the colonic barrier function.

Dietary inclusion of a bacterial meal has recently been shown to efficiently abolish soybean meal-induced enteritis in Atlantic salmon. The objective of this study was to investigate whether inclusion of this bacterial meal in the diet could abrogate disease development in a murine model of epithelial injury and colitis and thus possibly have therapeutic potential in human inflammatory bowel disease. C57BL/6N mice were fed ad libitum a control diet or an experimental diet containing 254 g/kg of body weight BioProtein, a bacterial meal consisting of Methylococcus capsulatus (Bath), together with the heterogenic bacteria Ralstonia sp., Brevibacillus agri, and Aneurinibacillus sp. At day 8, colitis was induced by 3.5% dextran sulfate sodium (DSS) ad libitum in the drinking water for 6 days. Symptoms of DSS treatment were less profound after prophylactic treatment with the diet containing the BioProtein. Colitis-associated parameters such as reduced body weight, colon shortening, and epithelial damage also showed significant improvement. Levels of acute-phase reactants, proteins whose plasma concentrations increase in response to inflammation, and neutrophil infiltration were reduced. On the other, increased epithelial cell proliferation and enhanced mucin 2 (Muc2) transcription indicated improved integrity of the colonic epithelial layer. BioProtein mainly consists of Methylococcus capsulatus (Bath) (88%). The results that we obtained when using a bacterial meal consisting of M. capsulatus (Bath) were similar to those obtained when using BioProtein in the DSS model. Our results show that a bacterial meal of the noncommensal bacterium M. capsulatus (Bath) has the potential to attenuate DSS-induced colitis in mice by enhancing colonic barrier function, as judged by increased epithelial proliferation and increased Muc2 transcription.

The deletion of YLR042c improves ethanolic xylose fermentation by recombinant Saccharomyces cerevisiae.

In a recent study combining transcriptome analyses of a number of recombinant laboratory and industrial S. cerevisiae strains with improved xylose utilization and their respective control strains, the ORF YLR042c was identified as a downregulated gene and it was shown that the gene deletion improved aerobic growth on xylose in the tested strain background. In the present study, the influence of deleting YLR042c on xylose fermentation was investigated in two different xylose-fermenting strains: TMB3001, which expresses genes from the initial xylose catabolizing pathway, including heterologous xylose reductase (XR) and xylitol dehydrogenase (XDH) and endogenous xylulokinase (XK); and TMB3057, which, in addition to the initial xylose catabolizing pathway, overexpresses the endogenous genes encoding the non-oxidative pentose phosphate pathway enzymes. The deletion of YLR042c led to improved aerobic growth on xylose in both strain backgrounds. However, the effect was more significant in the strain with the poorer growth rate on xylose (TMB3001). Under anaerobic conditions, the deletion of YLR042c increased the specific xylose consumption rate and the ethanol and xylitol yields. In strain TMB3057, xylose consumption was also improved at low concentrations and during co-fermentation of xylose and glucose. The effect of the gene deletion and overexpression was also tested for different carbon sources. Altogether, these results suggest that YLR042c influences xylose and the assimilation of carbon sources other than glucose, and that the effect could be at the level of sugar transport or sugar signalling.

Improved xylose and arabinose utilization by an industrial recombinant Saccharomyces cerevisiae strain using evolutionary engineering.

BACKGROUND: Cost-effective fermentation of lignocellulosic hydrolysate to ethanol by Saccharomyces cerevisiae requires efficient mixed sugar utilization. Notably, the rate and yield of xylose and arabinose co-fermentation to ethanol must be enhanced. RESULTS: Evolutionary engineering was used to improve the simultaneous conversion of xylose and arabinose to ethanol in a recombinant industrial Saccharomyces cerevisiae strain carrying the heterologous genes for xylose and arabinose utilization pathways integrated in the genome. The evolved strain TMB3130 displayed an increased consumption rate of xylose and arabinose under aerobic and anaerobic conditions. Improved anaerobic ethanol production was achieved at the expense of xylitol and glycerol but arabinose was almost stoichiometrically converted to arabitol. Further characterization of the strain indicated that the selection pressure during prolonged continuous culture in xylose and arabinose medium resulted in the improved transport of xylose and arabinose as well as increased levels of the enzymes from the introduced fungal xylose pathway. No mutation was found in any of the genes from the pentose converting pathways. CONCLUSION: To the best of our knowledge, this is the first report that characterizes the molecular mechanisms for improved mixed-pentose utilization obtained by evolutionary engineering of a recombinant S. cerevisiae strain. Increased transport of pentoses and increased activities of xylose converting enzymes contributed to the improved phenotype.

Arabinose and xylose fermentation by recombinant Saccharomyces cerevisiae expressing a fungal pentose utilization pathway.

BACKGROUND: Sustainable and economically viable manufacturing of bioethanol from lignocellulose raw material is dependent on the availability of a robust ethanol producing microorganism, able to ferment all sugars present in the feedstock, including the pentose sugars L-arabinose and D-xylose. Saccharomyces cerevisiae is a robust ethanol producer, but needs to be engineered to achieve pentose sugar fermentation. RESULTS: A new recombinant S. cerevisiae strain expressing an improved fungal pathway for the utilization of L-arabinose and D-xylose was constructed and characterized. The new strain grew aerobically on L-arabinose and D-xylose as sole carbon sources. The activities of the enzymes constituting the pentose utilization pathway(s) and product formation during anaerobic mixed sugar fermentation were characterized. CONCLUSION: Pentose fermenting recombinant S. cerevisiae strains were obtained by the expression of a pentose utilization pathway of entirely fungal origin. During anaerobic fermentation the strain produced biomass and ethanol. L-arabitol yield was 0.48 g per gram of consumed pentose sugar, which is considerably less than previously reported for D-xylose reductase expressing strains co-fermenting L-arabinose and D-xylose, and the xylitol yield was 0.07 g per gram of consumed pentose sugar.

Xylose reductase from Pichia stipitis with altered coenzyme preference improves ethanolic xylose fermentation by recombinant Saccharomyces cerevisiae.

BACKGROUND: Xylose reductase (XR) and xylitol dehydrogenase (XDH) from Pichia stipitis are the two enzymes most commonly used in recombinant Saccharomyces cerevisiae strains engineered for xylose utilization. The availability of NAD+ for XDH is limited during anaerobic xylose fermentation because of the preference of XR for NADPH. This in turn results in xylitol formation and reduced ethanol yield. The coenzyme preference of P. stipitis XR was changed by site-directed mutagenesis with the aim to engineer it towards NADH-preference. RESULTS: XR variants were evaluated in S. cerevisiae strains with the following genetic modifications: overexpressed native P. stipitis XDH, overexpressed xylulokinase, overexpressed non-oxidative pentose phosphate pathway and deleted GRE3 gene encoding an NADPH dependent aldose reductase. All overexpressed genes were chromosomally integrated to ensure stable expression. Crude extracts of four different strains overexpressing genes encoding native P. stipitis XR, K270M and K270R mutants, as well as Candida parapsilosis XR, were enzymatically characterized. The physiological effects of the mutations were investigated in anaerobic xylose fermentation. The strain overexpressing P. stipitis XR with the K270R mutation gave an ethanol yield of 0.39 g (g consumed sugars)-1, a xylitol yield of 0.05 g (g consumed xylose)-1 and a xylose consumption rate of 0.28 g (g biomass)-1 h-1 in continuous fermentation at a dilution rate of 0.12 h-1, with 10 g l-1 glucose and 10 g l-1 xylose as carbon sources. CONCLUSION: The cofactor preference of P. stipitis XR was altered by site-directed mutagenesis. When the K270R XR was combined with a metabolic engineering strategy that ensures high xylose utilization capabilities, a recombinant S. cerevisiae strain was created that provides a unique combination of high xylose consumption rate, high ethanol yield and low xylitol yield during ethanolic xylose fermentation.

Screening of Saccharomyces cerevisiae strains with respect to anaerobic growth in non-detoxified lignocellulose hydrolysate.

A microplate screening method was used to assess anaerobic growth of 12 Saccharomyces cerevisiae strains in barley straw, spruce and wheat straw hydrolysate. The assay demonstrated significant differences in inhibitor tolerance among the strains. In addition, growth inhibition by the three hydrolysates differed so that wheat hydrolysate supported growth up to 70%, while barley hydrolysate only supported growth up to 50%, with dilute-acid spruce hydrolysate taking an intermediate position.

Identification of common traits in improved xylose-growing Saccharomyces cerevisiae for inverse metabolic engineering.

Four recombinant Saccharomyces cerevisiae strains with enhanced xylose growth (TMB3400, C1, C5 and BH42) were compared with two control strains (TMB3399, TMB3001) through genome-wide transcription analysis in order to identify novel targets for inverse metabolic engineering. A subset of 13 genes with changed expression levels in all improved strains was selected for further analysis. Thirteen validation strains and two reference strains were constructed to investigate the effect of overexpressing or deleting these genes in xylose-utilizing S. cerevisiae. Improved aerobic growth rates on xylose were observed in five cases. The strains overexpressing SOL3 and TAL1 grew 19% and 24% faster than their reference strain, and the strains carrying deletions of YLR042C, MNI1 or RPA49 grew 173%, 62% and 90% faster than their reference strain.

Simultaneous saccharification and co-fermentation of glucose and xylose in steam-pretreated corn stover at high fiber content with Saccharomyces cerevisiae TMB3400.

The two main sugars in the agricultural by-product corn stover are glucose and xylose. Co-fermentation of glucose and xylose at high content of water-insoluble solids (WIS) without detoxification is a prerequisite to obtain high ethanol concentration and to reduce production costs. A recombinant strain of Saccharomyces cerevisiae, TMB3400, was used in simultaneous saccharification and fermentation (SSF) of whole pretreated slurry of corn stover at high WIS. TMB3400 co-fermented glucose and xylose with relatively high ethanol yields giving high final ethanol concentration. The ethanol productivity increased with increasing concentration of pretreatment hydrolysate in the yeast production medium and when SSF was performed in a fed-batch mode.

The expression of a Pichia stipitis xylose reductase mutant with higher K(M) for NADPH increases ethanol production from xylose in recombinant Saccharomyces cerevisiae.

Xylose fermentation by Saccharomyces cerevisiae requires the introduction of a xylose pathway, either similar to that found in the natural xylose-utilizing yeasts Pichia stipitis and Candida shehatae or similar to the bacterial pathway. The use of NAD(P)H-dependent XR and NAD(+)-dependent XDH from P. stipitis creates a cofactor imbalance resulting in xylitol formation. The effect of replacing the native P. stipitis XR with a mutated XR with increased K(M) for NADPH was investigated for xylose fermentation to ethanol by recombinant S. cerevisiae strains. Enhanced ethanol yields accompanied by decreased xylitol yields were obtained in strains carrying the mutated XR. Flux analysis showed that strains harboring the mutated XR utilized a larger fraction of NADH for xylose reduction. The overproduction of the mutated XR resulted in an ethanol yield of 0.40 g per gram of sugar and a xylose consumption rate of 0.16 g per gram of biomass per hour in chemostat culture (0.06/h) with 10 g/L glucose and 10 g/L xylose as carbon source.