Succession of physiological stages hallmarks the transcriptomic response of the fungus Aspergillus niger to lignocellulose.

BACKGROUND: Understanding how fungi degrade lignocellulose is a cornerstone of improving renewables-based biotechnology, in particular for the production of hydrolytic enzymes. Considerable progress has been made in investigating fungal degradation during time-points where CAZyme expression peaks. However, a robust understanding of the fungal survival strategies over its life time on lignocellulose is thereby missed. Here we aimed to uncover the physiological responses of the biotechnological workhorse and enzyme producer Aspergillus niger over its life time to six substrates important for biofuel production. RESULTS: We analysed the response of A. niger to the feedstock Miscanthus and compared it with our previous study on wheat straw, alone or in combination with hydrothermal or ionic liquid feedstock pretreatments. Conserved (substrate-independent) metabolic responses as well as those affected by pretreatment and feedstock were identified via multivariate analysis of genome-wide transcriptomics combined with targeted transcript and protein analyses and mapping to a metabolic model. Initial exposure to all substrates increased fatty acid beta-oxidation and lipid metabolism transcripts. In a strain carrying a deletion of the ortholog of the Aspergillus nidulans fatty acid beta-oxidation transcriptional regulator farA, there was a reduction in expression of selected lignocellulose degradative CAZyme-encoding genes suggesting that beta-oxidation contributes to adaptation to lignocellulose. Mannan degradation expression was wheat straw feedstock-dependent and pectin degradation was higher on the untreated substrates. In the later life stages, known and novel secondary metabolite gene clusters were activated, which are of high interest due to their potential to synthesize bioactive compounds. CONCLUSION: In this study, which includes the first transcriptional response of Aspergilli to Miscanthus, we highlighted that life time as well as substrate composition and structure (via variations in pretreatment and feedstock) influence the fungal responses to lignocellulose. We also demonstrated that the fungal response contains physiological stages that are conserved across substrates and are typically found outside of the conditions with high CAZyme expression, as exemplified by the stages that are dominated by lipid and secondary metabolism.

The establishment of a marine focused biorefinery for bioethanol production using seawater and a novel marine yeast strain.

Current technologies for bioethanol production rely on the use of freshwater for preparing the fermentation media and use yeasts of a terrestrial origin. Life cycle assessment has suggested that between 1,388 to 9,812 litres of freshwater are consumed for every litre of bioethanol produced. Hence, bioethanol is considered a product with a high-water footprint. This paper investigated the use of seawater-based media and a novel marine yeast strain 'Saccharomyces cerevisiae AZ65' to reduce the water footprint of bioethanol. Results revealed that S. cerevisiae AZ65 had a significantly higher osmotic tolerance when compared with the terrestrial reference strain. Using 15-L bioreactors, S. cerevisiae AZ65 produced 93.50 g/L ethanol with a yield of 83.33% (of the theoretical yield) and a maximum productivity of 2.49 g/L/h when using seawater-YPD media. This approach was successfully applied using an industrial fermentation substrate (sugarcane molasses). S. cerevisiae AZ65 produced 52.23 g/L ethanol using molasses media prepared in seawater with a yield of 73.80% (of the theoretical yield) and a maximum productivity of 1.43 g/L/h. These results demonstrated that seawater can substitute freshwater for bioethanol production without compromising production efficiency. Results also revealed that marine yeast is a potential candidate for use in the bioethanol industry especially when using seawater or high salt based fermentation media.

Expression of Aspergillus niger CAZymes is determined by compositional changes in wheat straw generated by hydrothermal or ionic liquid pretreatments.

BACKGROUND: The capacity of fungi, such as Aspergillus niger, to degrade lignocellulose is harnessed in biotechnology to generate biofuels and high-value compounds from renewable feedstocks. Most feedstocks are currently pretreated to increase enzymatic digestibility: improving our understanding of the transcriptomic responses of fungi to pretreated lignocellulosic substrates could help to improve the mix of activities and reduce the production costs of commercial lignocellulose saccharifying cocktails. RESULTS: We investigated the responses of A. niger to untreated, ionic liquid and hydrothermally pretreated wheat straw over a 5-day time course using RNA-seq and targeted proteomics. The ionic liquid pretreatment altered the cellulose crystallinity while retaining more of the hemicellulosic sugars than the hydrothermal pretreatment. Ionic liquid pretreatment of straw led to a dynamic induction and repression of genes, which was correlated with the higher levels of pentose sugars saccharified from the ionic liquid-pretreated straw. Hydrothermal pretreatment of straw led to reduced levels of transcripts of genes encoding carbohydrate-active enzymes as well as the derived proteins and enzyme activities. Both pretreatments abolished the expression of a large set of genes encoding pectinolytic enzymes. These reduced levels could be explained by the removal of parts of the lignocellulose by the hydrothermal pretreatment. The time course also facilitated identification of temporally limited gene induction patterns. CONCLUSIONS: The presented transcriptomic and biochemical datasets demonstrate that pretreatments caused modifications of the lignocellulose, to both specific structural features as well as the organisation of the overall lignocellulosic structure, that determined A. niger transcript levels. The experimental setup allowed reliable detection of substrate-specific gene expression patterns as well as hitherto non-expressed genes. Our data suggest beneficial effects of using untreated and IL-pretreated straw, but not HT-pretreated straw, as feedstock for CAZyme production.

The effect of adenosine monophosphate deaminase overexpression on the accumulation of umami-related metabolites in tomatoes.

KEY MESSAGE: This study highlights the changes in umami-related nucleotide and glutamate levels when the AMP deaminase gene was elevated in transgenic tomato. Taste is perceived as one of a combination of five sensations, sweet, sour, bitter, salty, and umami. The umami taste is best known as a savoury sensation and plays a central role in food flavour, palatability, and eating satisfaction. Umami flavour can be imparted by the presence of glutamate and is greatly enhanced by the addition of ribonucleotides, such as inosine monophosphate (IMP) and guanosine monophosphate (GMP). The production of IMP is regulated by the enzyme adenosine monophosphate (AMP) deaminase which functions to convert AMP into IMP. We have generated transgenic tomato (Solanum lycopersicum) lines over expressing AMP deaminase under the control of a fruit-specific promoter. The transgenic lines showed substantially enhanced levels of AMP deaminase expression in comparison to the wild-type control. Elevated AMP deaminase levels resulted in the reduced accumulation of glutamate and increased levels of the umami nucleotide GMP. AMP concentrations were unchanged. The effects on the levels of glutamate and GMP were unexpected and are discussed in relation to the metabolite flux within this pathway.

Genetic improvement of tomato by targeted control of fruit softening.

Controlling the rate of softening to extend shelf life was a key target for researchers engineering genetically modified (GM) tomatoes in the 1990s, but only modest improvements were achieved. Hybrids grown nowadays contain 'non-ripening mutations' that slow ripening and improve shelf life, but adversely affect flavor and color. We report substantial, targeted control of tomato softening, without affecting other aspects of ripening, by silencing a gene encoding a pectate lyase.

Expression of Mitochondrial Cytochrome C Oxidase Chaperone Gene (COX20) Improves Tolerance to Weak Acid and Oxidative Stress during Yeast Fermentation.

INTRODUCTION: Saccharomyces cerevisiae is the micro-organism of choice for the conversion of fermentable sugars released by the pre-treatment of lignocellulosic material into bioethanol. Pre-treatment of lignocellulosic material releases acetic acid and previous work identified a cytochrome oxidase chaperone gene (COX20) which was significantly up-regulated in yeast cells in the presence of acetic acid. RESULTS: A Δcox20 strain was sensitive to the presence of acetic acid compared with the background strain. Overexpressing COX20 using a tetracycline-regulatable expression vector system in a Δcox20 strain, resulted in tolerance to the presence of acetic acid and tolerance could be ablated with addition of tetracycline. Assays also revealed that overexpression improved tolerance to the presence of hydrogen peroxide-induced oxidative stress. CONCLUSION: This is a study which has utilised tetracycline-regulated protein expression in a fermentation system, which was characterised by improved (or enhanced) tolerance to acetic acid and oxidative stress.

Authentication of processed meat products by peptidomic analysis using rapid ambient mass spectrometry.

We present the application of a novel ambient LESA-MS method for the authentication of processed meat products. A set of 25 species and protein-specific heat stable peptide markers has been detected in processed samples manufactured from beef, pork, horse, chicken and turkey meat. We demonstrate that several peptides derived from myofibrillar and sarcoplasmic proteins are sufficiently resistant to processing to serve as specific markers of processed products. The LESA-MS technique required minimal sample preparation without fractionation and enabled the unambiguous and simultaneous identification of skeletal muscle proteins and peptides as well as other components of animal origin, including the milk protein such as casein alpha-S1, in whole meat product digests. We have identified, for the first time, six fast type II and five slow/cardiac type I MHC peptide markers in various processed meat products. The study demonstrates that complex mixtures of processed proteins/peptides can be examined effectively using this approach.

Rapid detection of peptide markers for authentication purposes in raw and cooked meat using ambient liquid extraction surface analysis mass spectrometry.

In this Article, our previously developed ambient LESA-MS methodology is implemented to analyze five types of thermally treated meat species, namely, beef, pork, horse, chicken, and turkey meat, to select and identify heat-stable and species-specific peptide markers. In-solution tryptic digests of cooked meats were deposited onto a polymer surface, followed by LESA-MS analysis and evaluation using multivariate data analysis and tandem electrospray MS. The five types of cooked meat were clearly discriminated using principal component analysis and orthogonal partial least-squares discriminant analysis. 23 heat stable peptide markers unique to species and muscle protein were identified following data-dependent tandem LESA-MS analysis. Surface extraction and direct ambient MS analysis of mixtures of cooked meat species was performed for the first time and enabled detection of 10% (w/w) of pork, horse, and turkey meat and 5% (w/w) of chicken meat in beef, using the developed LESA-MS/MS analysis. The study shows, for the first time, that ambient LESA-MS methodology displays specificity sufficient to be implemented effectively for the analysis of processed and complex peptide digests. The proposed approach is much faster and simpler than other measurement tools for meat speciation; it has potential for application in other areas of meat science or food production.

Comparison of SNP-based detection assays for food analysis: Coffee authentication.

Recently, DNA-based authentication methods were developed to serve as complementary approaches to analytical chemistry techniques. The single nucleotide polymorphism (SNP)-based reaction chemistries, when combined with the existing detection methods, could result in numerous analytical approaches, all with particular advantages and disadvantages. The dual aim of this study was (a) to develop SNP-based analytical assays such as the single-base primer extension (SNaPShot) and pyrosequencing in order to differentiate Arabica and Robusta varieties for the authentication of coffee beans and (b) to compare the performances of SNaPshot, pyrosequencing and the previously developed polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) using an Agilent 2100 Bioanalyzer on the basis of linearity (R2) and LOD, expressed as percentage of the adulterant species, using green coffee beans (Arabica and Robusta) as a food model. The results showed that SNaPshot analysis exhibited the best LOD, whereas pyrosequencing revealed the best linearity (R2 = 0.997). The PCR-RFLP assay using the Agilent 2100 Bioanalyzer could prove to be a very useful method for a laboratory that lacks sequencing facilities but it can be used only if a SNP creates/deletes a restriction site.

The genetic basis of variation in clean lineages of Saccharomyces cerevisiae in response to stresses encountered during bioethanol fermentations.

Saccharomyces cerevisiae is the micro-organism of choice for the conversion of monomeric sugars into bioethanol. Industrial bioethanol fermentations are intrinsically stressful environments for yeast and the adaptive protective response varies between strain backgrounds. With the aim of identifying quantitative trait loci (QTL's) that regulate phenotypic variation, linkage analysis on six F1 crosses from four highly divergent clean lineages of S. cerevisiae was performed. Segregants from each cross were assessed for tolerance to a range of stresses encountered during industrial bioethanol fermentations. Tolerance levels within populations of F1 segregants to stress conditions differed and displayed transgressive variation. Linkage analysis resulted in the identification of QTL's for tolerance to weak acid and osmotic stress. We tested candidate genes within loci identified by QTL using reciprocal hemizygosity analysis to ascertain their contribution to the observed phenotypic variation; this approach validated a gene (COX20) for weak acid stress and a gene (RCK2) for osmotic stress. Hemizygous transformants with a sensitive phenotype carried a COX20 allele from a weak acid sensitive parent with an alteration in its protein coding compared with other S. cerevisiae strains. RCK2 alleles reveal peptide differences between parental strains and the importance of these changes is currently being ascertained.

Marine yeast isolation and industrial application.

Over the last century, terrestrial yeasts have been widely used in various industries, such as baking, brewing, wine, bioethanol and pharmaceutical protein production. However, only little attention has been given to marine yeasts. Recent research showed that marine yeasts have several unique and promising features over the terrestrial yeasts, for example higher osmosis tolerance, higher special chemical productivity and production of industrial enzymes. These indicate that marine yeasts have great potential to be applied in various industries. This review gathers the most recent techniques used for marine yeast isolation as well as the latest applications of marine yeast in bioethanol, pharmaceutical and enzyme production fields.

Tryptic digestion coupled with ambient desorption electrospray ionization and liquid extraction surface analysis mass spectrometry enabling identification of skeletal muscle proteins in mixtures and distinguishing between beef, pork, horse, chicken, and turkey meat.

The use of ambient desorption electrospray ionization mass spectrometry (DESI-MS) and liquid extraction surface analysis mass spectrometry (LESA-MS) is explored for the first time to analyze skeletal muscle proteins obtained from a mixture of standard proteins and raw meat. Single proteins and mixtures of up to five proteins (myoglobin, troponin C, actin, bovine serum albumin (BSA), tropomyosin) were deposited onto a polymer surface, followed by in situ tryptic digestion and comparative analysis using DESI-MS and LESA-MS using tandem electrospray MS. Peptide peaks specific to individual proteins were readily distinguishable with good signal-to-noise ratio in the five-component mixture. LESA-MS gave a more stable analysis and greater sensitivity compared with DESI-MS. Meat tryptic digests were subjected to peptidomics analysis by DESI-MS and LESA-MS. Bovine, horse, pig, chicken, and turkey muscle digests were clearly discriminated using multivariate data analysis (MVA) of the peptidomic data sets. The most abundant skeletal muscle proteins were identified and correctly classified according to the species following MS/MS analysis. The study shows, for the first time, that ambient ionization techniques such as DESI-MS and LESA-MS have great potential for species-specific analysis and differentiation of skeletal muscle proteins by direct surface desorption.

Phenotypic characterisation of Saccharomyces spp. yeast for tolerance to stresses encountered during fermentation of lignocellulosic residues to produce bioethanol.

BACKGROUND: During industrial fermentation of lignocellulose residues to produce bioethanol, microorganisms are exposed to a number of factors that influence productivity. These include inhibitory compounds produced by the pre-treatment processes required to release constituent carbohydrates from biomass feed-stocks and during fermentation, exposure of the organisms to stressful conditions. In addition, for lignocellulosic bioethanol production, conversion of both pentose and hexose sugars is a pre-requisite for fermentative organisms for efficient and complete conversion. All these factors are important to maximise industrial efficiency, productivity and profit margins in order to make second-generation bioethanol an economically viable alternative to fossil fuels for future transport needs. RESULTS: The aim of the current study was to assess Saccharomyces yeasts for their capacity to tolerate osmotic, temperature and ethanol stresses and inhibitors that might typically be released during steam explosion of wheat straw. Phenotypic microarray analysis was used to measure tolerance as a function of growth and metabolic activity. Saccharomyces strains analysed in this study displayed natural variation to each stress condition common in bioethanol fermentations. In addition, many strains displayed tolerance to more than one stress, such as inhibitor tolerance combined with fermentation stresses. CONCLUSIONS: Our results suggest that this study could identify a potential candidate strain or strains for efficient second generation bioethanol production. Knowledge of the Saccharomyces spp. strains grown in these conditions will aid the development of breeding programmes in order to generate more efficient strains for industrial fermentations.

RNA-sequencing reveals the complexities of the transcriptional response to lignocellulosic biofuel substrates in Aspergillus niger.

BACKGROUND: Saprobic fungi are the predominant industrial sources of Carbohydrate Active enZymes (CAZymes) used for the saccharification of lignocellulose during the production of second generation biofuels. The production of more effective enzyme cocktails is a key objective for efficient biofuel production. To achieve this objective, it is crucial to understand the response of fungi to lignocellulose substrates. Our previous study used RNA-seq to identify the genes induced in Aspergillus niger in response to wheat straw, a biofuel feedstock, and showed that the range of genes induced was greater than previously seen with simple inducers. RESULTS: In this work we used RNA-seq to identify the genes induced in A. niger in response to short rotation coppice willow and compared this with the response to wheat straw from our previous study, at the same time-point. The response to willow showed a large increase in expression of genes encoding CAZymes. Genes encoding the major activities required to saccharify lignocellulose were induced on willow such as endoglucanases, cellobiohydrolases and xylanases. The transcriptome response to willow had many similarities with the response to straw with some significant differences in the expression levels of individual genes which are discussed in relation to differences in substrate composition or other factors. Differences in transcript levels include higher levels on wheat straw from genes encoding enzymes classified as members of GH62 (an arabinofuranosidase) and CE1 (a feruloyl esterase) CAZy families whereas two genes encoding endoglucanases classified as members of the GH5 family had higher transcript levels when exposed to willow. There were changes in the cocktail of enzymes secreted by A. niger when cultured with willow or straw. Assays for particular enzymes as well as saccharification assays were used to compare the enzyme activities of the cocktails. Wheat straw induced an enzyme cocktail that saccharified wheat straw to a greater extent than willow. Genes not encoding CAZymes were also induced on willow such as hydrophobins as well as genes of unknown function. Several genes were identified as promising targets for future study. CONCLUSIONS: By comparing this first study of the global transcriptional response of a fungus to willow with the response to straw, we have shown that the inducing lignocellulosic substrate has a marked effect upon the range of transcripts and enzymes expressed by A. niger. The use by industry of complex substrates such as wheat straw or willow could benefit efficient biofuel production.

RNA-sequencing reveals the complexities of the transcriptional response to lignocellulosic biofuel substrates in Aspergillus niger.

BACKGROUND: Saprobic fungi are the predominant industrial sources of Carbohydrate Active enZymes (CAZymes) used for the saccharification of lignocellulose during the production of second generation biofuels. The production of more effective enzyme cocktails is a key objective for efficient biofuel production. To achieve this objective, it is crucial to understand the response of fungi to lignocellulose substrates. Our previous study used RNA-seq to identify the genes induced in Aspergillus niger in response to wheat straw, a biofuel feedstock, and showed that the range of genes induced was greater than previously seen with simple inducers. RESULTS: In this work we used RNA-seq to identify the genes induced in A. niger in response to short rotation coppice willow and compared this with the response to wheat straw from our previous study, at the same time-point. The response to willow showed a large increase in expression of genes encoding CAZymes. Genes encoding the major activities required to saccharify lignocellulose were induced on willow such as endoglucanases, cellobiohydrolases and xylanases. The transcriptome response to willow had many similarities with the response to straw with some significant differences in the expression levels of individual genes which are discussed in relation to differences in substrate composition or other factors. Differences in transcript levels include higher levels on wheat straw from genes encoding enzymes classified as members of GH62 (an arabinofuranosidase) and CE1 (a feruloyl esterase) CAZy families whereas two genes encoding endoglucanases classified as members of the GH5 family had higher transcript levels when exposed to willow. There were changes in the cocktail of enzymes secreted by A. niger when cultured with willow or straw. Assays for particular enzymes as well as saccharification assays were used to compare the enzyme activities of the cocktails. Wheat straw induced an enzyme cocktail that saccharified wheat straw to a greater extent than willow. Genes not encoding CAZymes were also induced on willow such as hydrophobins as well as genes of unknown function. Several genes were identified as promising targets for future study. CONCLUSIONS: By comparing this first study of the global transcriptional response of a fungus to willow with the response to straw, we have shown that the inducing lignocellulosic substrate has a marked effect upon the range of transcripts and enzymes expressed by A. niger. The use by industry of complex substrates such as wheat straw or willow could benefit efficient biofuel production.

Null mutants of individual RABA genes impact the proportion of different cell wall components in stem tissue of Arabidopsis thaliana.

In Arabidopsis, and other plants, the RABA GTPases (orthologous to the Rab11a of mammals) have expanded in number and diversity and have been shown to belong to eight sub clades, some of which have been implicated in controlling vesicles that traffic cell wall polymers and enzymes that synthesise or modify them to the cell wall. In order to investigate this, we have investigated whether T-DNA insertion knockouts of individual RABA genes belonging to different sub clades, impact on the composition of the plant cell wall. Single gene knockouts of the RABA1, RABA2 and RABA4 sub clades primarily affected the percentage composition of pectin, cellulose and hemicellulose within the cell wall, respectively, despite having no obvious phenotype in the whole plant. We hypothesise that vesicles carrying specific types of cargoes from the Golgi to the cell surface may be regulated by particular sub types of RABA proteins, a finding that could have wider implications for how trafficking systems work and could be a useful tool in cell wall research and other fields of plant biology.

Cell wall composition of tomato fruit changes during development and inhibition of vesicle trafficking is associated with reduced pectin levels and reduced softening.

Fruit development entails a multitude of biochemical changes leading up to the mature green stage. During this period the cell wall will undergo complex compositional and structural changes. Inhibition of genes encoding elements of the machinery involved in trafficking to the cell wall presents us with a useful tool to study these changes and their associated phenotypes. An antisense SlRab11a transgene has previously been shown to reduce ripening-associated fruit softening. SlRab11a is highly expressed during fruit development which is associated with a period of pectin influx into the wall. We have analysed the cell wall polysaccharides at different stages of growth and ripening of wild type and antisense SlRab11a transgenic tomato (Solanum lycopersicum cv, Ailsa Craig) fruit. Our results demonstrated intriguing changes in cell wall composition during the development and ripening of wild type Alisa Craig tomato fruit. Analysis of SlRab11a expression by TaqMan PCR showed it to be expressed most strongly during growth of the fruit, suggesting a possible role in cell wall deposition. The SlRab11a antisense fruit had a decreased proportion of pectin in the cell wall compared with the wild type. We suggest a new approach for modification of fruit shelf-life by changing cell wall deposition rather than cell wall hydrolytic enzymes.

Quality parameters and antioxidant properties in organic and conventionally grown broccoli after pre-storage hot water treatment.

BACKGROUND: Demand for broccoli has increased due to its high content of bioactive compounds. However, broccoli is a perishable commodity with a short shelf life mainly due to dehydration, yellowing and losses of bioactive compounds. Thus, efficient treatments to preserve broccoli quality are needed. RESULTS: The effect of heat treatment on senescence and antioxidant compounds evolution during storage at 20 °C was evaluated in organic and conventionally grown broccoli. Senescence evolved quickly as manifested by floral head yellowing, which was higher in conventional than in organic broccolis, but senescence was significantly delayed by heat treatment. All organic acids, including ascorbic acid, were found at higher concentrations in organic than in conventional broccoli at harvest but decreased during storage in all broccolis. Phenolic concentration and antioxidant activity (in both hydrophilic and lipophilic fractions) also decreased during storage, although these decreases were higher in conventional than in organic broccolis, and no differences were found attributable to heat treatment. CONCLUSIONS: Heat treatment was effective in delaying broccoli senescence, manifested by chlorophyll retention. In addition, organic broccoli maintained higher concentrations of bioactive compounds (ascorbic acid and phenolics) and antioxidant potential during storage than conventional broccoli, with higher potential health beneficial effects.

Uncovering the genome-wide transcriptional responses of the filamentous fungus Aspergillus niger to lignocellulose using RNA sequencing.

A key challenge in the production of second generation biofuels is the conversion of lignocellulosic substrates into fermentable sugars. Enzymes, particularly those from fungi, are a central part of this process, and many have been isolated and characterised. However, relatively little is known of how fungi respond to lignocellulose and produce the enzymes necessary for dis-assembly of plant biomass. We studied the physiological response of the fungus Aspergillus niger when exposed to wheat straw as a model lignocellulosic substrate. Using RNA sequencing we showed that, 24 hours after exposure to straw, gene expression of known and presumptive plant cell wall-degrading enzymes represents a huge investment for the cells (about 20% of the total mRNA). Our results also uncovered new esterases and surface interacting proteins that might form part of the fungal arsenal of enzymes for the degradation of plant biomass. Using transcription factor deletion mutants (xlnR and creA) to study the response to both lignocellulosic substrates and low carbon source concentrations, we showed that a subset of genes coding for degradative enzymes is induced by starvation. Our data support a model whereby this subset of enzymes plays a scouting role under starvation conditions, testing for available complex polysaccharides and liberating inducing sugars, that triggers the subsequent induction of the majority of hydrolases. We also showed that antisense transcripts are abundant and that their expression can be regulated by growth conditions.