Complex carbohydrates of plants are the main food sources of animals and microbes, and serve as promising renewable feedstock for biofuel and biomaterial production. Carbohydrate active enzymes (CAZymes) are the most important enzymes for complex carbohydrate metabolism. With an increasing number of plant and plant-associated microbial genomes and metagenomes being sequenced, there is an urgent need of automatic tools for genomic data mining of CAZymes. We developed the dbCAN web server in 2012 to provide a public service for automated CAZyme annotation for newly sequenced genomes. Here, dbCAN2 (http://cys.bios.niu.edu/dbCAN2) is presented as an updated meta server, which integrates three state-of-the-art tools for CAZome (all CAZymes of a genome) annotation: (i) HMMER search against the dbCAN HMM (hidden Markov model) database; (ii) DIAMOND search against the CAZy pre-annotated CAZyme sequence database and (iii) Hotpep search against the conserved CAZyme short peptide database. Combining the three outputs and removing CAZymes found by only one tool can significantly improve the CAZome annotation accuracy. In addition, dbCAN2 now also accepts nucleotide sequence submission, and offers the service to predict physically linked CAZyme gene clusters (CGCs), which will be a very useful online tool for identifying putative polysaccharide utilization loci (PULs) in microbial genomes or metagenomes.

Transgenic cassava (Manihot esculenta Crantz, cv MCol22) plants with a 92% reduction in cyanogenic glucoside content in tubers and acyanogenic (,1% of wild type) leaves were obtained by RNA interference to block expression of CYP79D1 and CYP79D2, the two paralogous genes encoding the first committed enzymes in linamarin and lotaustralin synthesis. About 180 independent lines with acyanogenic (,1% of wild type) leaves were obtained. Only a few of these were depleted with respect to cyanogenic glucoside content in tubers. In agreement with this observation, girdling experiments demonstrated that cyanogenic glucosides are synthesized in the shoot apex and transported to the root, resulting in a negative concentration gradient basipetal in the plant with the concentration of cyanogenic glucosides being highest in the shoot apex and the petiole of the first unfolded leaf. Supply of nitrogen increased the cyanogenic glucoside concentration in the shoot apex. In situ polymerase chain reaction studies demonstrated that CYP79D1 and CYP79D2 were preferentially expressed in leaf mesophyll cells positioned adjacent to the epidermis. In young petioles, preferential expression was observed in the epidermis, in the two first cortex cell layers, and in the endodermis together with pericycle cells and specific parenchymatic cells around the laticifers. These data demonstrate that it is possible to drastically reduce the linamarin and lotaustralin content in cassava tubers by blockage of cyanogenic glucoside synthesis in leaves and petioles. The reduced flux to the roots of reduced nitrogen in the form of cyanogenic glucosides did not prevent tuber formation.Cassava (Manihot esculenta Crantz) is the most important root crop in the world and ranks second among African staple crops (Nweke et al., 2002). Cassava is vegetatively propagated through stem cuttings and produces well on poor soils. The tubers may be kept in the soil for extended time periods. This secures rural farmers a carbohydrate source in years with adverse growth conditions where other crops fail and famine would otherwise prevail. These features and high crop yield contribute to the importance of cassava in Africa, South East Asia, and South America Industrial cassava starch production is important, especially in South East Asia (Bokanga, 1994). Cassava leaves are not widely used as a food source despite their high protein content (Ngudi et al., 2003).Major drawbacks of the cassava crop are the low tuber protein content, rapid tuber perishability following harvest, and high content of the cyanogenic glucosides linamarin and lotaustralin in all tissues. Upon tissue disruption, the cyanogenic glucosides are brought in contact with b-glucosidases and a-hydroxynitrile lyases and degraded into cyanohydrins, hydrogen cyanide, and ketones (Conn, 1980). When cassava products are used as a primary staple food, careful processing to remove these toxic constituents is required to avoid chronic cyanide intoxication (Onabolu et al., 2002). Incomplete processing may result in hi...

BackgroundMicroRNAs are important regulators of gene expression at the post-transcriptional level and play an important role in many biological processes. Due to the important biological role it is of great interest to quantitatively determine their expression level in different biological settings.ResultsWe describe a PCR method for quantification of microRNAs based on a single reverse transcription reaction for all microRNAs combined with real-time PCR with two, microRNA-specific DNA primers. Primer annealing temperatures were optimized by adding a DNA tail to the primers and could be designed with a success rate of 94%. The method was able to quantify synthetic templates over eight orders of magnitude and readily discriminated between microRNAs with single nucleotide differences. Importantly, PCR with DNA primers yielded significantly higher amplification efficiencies of biological samples than a similar method based on locked nucleic acids-spiked primers, which is in agreement with the observation that locked nucleic acid interferes with efficient amplification of short templates. The higher amplification efficiency of DNA primers translates into higher sensitivity and precision in microRNA quantification.ConclusionsMiR-specific quantitative RT-PCR with DNA primers is a highly specific, sensitive and accurate method for microRNA quantification.

Discovery of keratin-degrading enzymes from fungi and bacteria has primarily focused on finding one protease with efficient keratinase activity. Recently, an investigation was conducted of all keratinases secreted from a fungus known to grow on keratinaceous materials, such as feather, horn, and hooves. The study demonstrated that a minimum of three keratinases is needed to break down keratin, an endo-acting, an exo-acting, and an oligopeptide-acting keratinase. Further, several studies have documented that disruption of sulfur bridges of the keratin structure acts synergistically with the keratinases to loosen the molecular structure, thus giving the enzymes access to their substrate, the protein structure. With such complexity, it is relevant to compare microbial keratin decomposition with the microbial decomposition of well-studied polymers such as cellulose and chitin. Interestingly, it was recently shown that the specialized enzymes, lytic polysaccharide monoxygenases (LPMOs), shown to be important for breaking the recalcitrance of cellulose and chitin, are also found in keratin-degrading fungi. A holistic view of the complex molecular self-assembling structure of keratin and knowledge about enzymatic and boosting factors needed for keratin breakdown have been used to formulate a hypothesis for mode of action of the LPMOs in keratin decomposition and for a model for degradation of keratin in nature. Testing such hypotheses and models still needs to be done. Even now, the hypothesis can serve as an inspiration for designing industrial processes for keratin decomposition for conversion of unexploited waste streams, chicken feather, and pig bristles into bioaccessible animal feed.

The content of the cyanogenic glucoside dhurrin in sorghum (Sorghum bicolor L. Moench) varies depending on plant age and growth conditions. The cyanide potential is highest shortly after onset of germination. At this stage, nitrogen application has no effect on dhurrin content, whereas in older plants, nitrogen application induces an increase. At all stages, the content of dhurrin correlates well with the activity of the two biosynthetic enzymes, CYP79A1 and CYP71E1, and with the protein and mRNA level for the two enzymes. During development, the activity of CYP79A1 is lower than the activity of CYP71E1, suggesting that CYP79A1 catalyzes the rate-limiting step in dhurrin synthesis as has previously been shown using etiolated seedlings. The site of dhurrin synthesis shifts from leaves to stem during plant development. In combination, the results demonstrate that dhurrin content in sorghum is largely determined by transcriptional regulation of the biosynthetic enzymes CYP79A1 and CYP71E1.More than 2,650 plant species from more than 550 genera and 130 families are known to be cyanogenic, and this phenomenon is typically based on the presence of cyanogenic glucosides (Hegnauer, 1986;Seigler, 1991). The biological function of cyanogenic glucosides has been difficult to assess (Jones, 1998;Selmar, 1999; Jones et al., 2000). Cyanogenic glucosides are constitutively produced in healthy plant tissues and belong to the class of natural products referred to as "phytoanticipins" (Osbourn, 1996). Mechanical disruption of plant tissue containing cyanogenic glucosides results in their degradation by the sequential action of ␤-glucosidases and ␣-hydroxynitrilases (Cicek and Esen, 1998; Lechtenberg and Nahrstedt, 1999;Selmar, 1999; Jones et al., 2000) and release of hydrogen cyanide. The toxicity of hydrogen cyanide renders it obvious to assume that cyanogenic glucosides repel herbivores (Jones, 1998). However, many trials do not support this hypothesis (Hruska, 1988) and effectiveness may be strongly influenced by the feeding strategy of the animals (Compton and Jones, 1985). With regard to the interaction between plants and microorganisms, the release of hydrogen cyanide from cyanogenic glucosides may be more damaging to the plant than to the microorganism because of inhibition of phytoalexin production (Lieberei et al., 1989). In accordance, highly cyanogenic plants are preferred by some fungi and insects compared with plants with lower cyanogenic potential (Nahrstedt, 1996; Møller and Seigler, 1999). Aglycones released from cyanogenic glucosides formed from Phe or Tyr may give rise to the formation of compounds with antifungal activities (Siebert et al., 1996). Other possible roles of cyanogenic glucosides are as nitrogen storage compounds (Clegg et al., 1979;Selmar et al., 1988; Forslund and Jonsson, 1997) or as osmoprotectants. The latter functions are likely to require the presence of high amounts of cyanogenic glucosides in a particular cell type or tissue. This is often the case as in sorghum (Sorghum bicolor), whe...

The first committed steps in the biosynthesis of the two cyanogenic glucosides linamarin and lotaustralin in cassava are the conversion of L-valine and L-isoleucine, respectively, to the corresponding oximes. Two fulllength cDNA clones that encode cytochromes P-450 catalyzing these reactions have been isolated. The two cassava cytochromes P-450 are 85% identical, share 54% sequence identity to CYP79A1 from sorghum, and have been assigned CYP79D1 and CYP79D2. Functional expression has been achieved using the methylotrophic yeast, Pichia pastoris. The amount of CYP79D1 isolated from 1 liter of P. pastoris culture exceeds the amounts that putatively could be isolated from 22,000 grown-up cassava plants. Each cytochrome P-450 metabolizes Lvaline as well as L-isoleucine consistent with the cooccurrence of linamarin and lotaustralin in cassava. CYP79D1 was isolated from P. pastoris. Reconstitution in lipid micelles showed that CYP79D1 has a higher k c value with L-valine as substrate than with L-isoleucine, which is consistent with linamarin being the major cyanogenic glucoside in cassava. Both CYP79D1 and CYP79D2 are present in the genome of cassava cultivar MCol22 in agreement with cassava being allotetraploid. CYP79D1 and CYP79D2 are actively transcribed, and production of acyanogenic cassava plants would therefore require down-regulation of both genes.

BackgroundMicroRNAs are small but biologically important RNA molecules. Although different methods can be used for quantification of microRNAs, quantitative PCR is regarded as the reference that is used to validate other methods. Several commercial qPCR assays are available but they often come at a high price and the sequences of the primers are not disclosed. An alternative to commercial assays is to manually design primers but this work is tedious and, hence, not practical for the design of primers for a larger number of targets.ResultsI have developed the software miRprimer for automatic design of primers for the method miR-specific RT-qPCR, which is one of the best performing microRNA qPCR methods available. The algorithm is based on an implementation of the previously published rules for manual design of miR-specific primers with the additional feature of evaluating the propensity of formation of secondary structures and primer dimers. Testing of the primers showed that 76 out of 79 primers (96%) worked for quantification of microRNAs by miR-specific RT-qPCR of mammalian RNA samples. This success rate corresponds to the success rate of manual primer design. Furthermore, primers designed by this method have been distributed to several labs and used successfully in published studies.ConclusionsThe software miRprimer is an automatic and easy method for design of functional primers for miR-specific RT-qPCR. The application is available as stand-alone software that will work on the MS Windows platform and in a developer version written in the Ruby programming language.

Functional prediction of carbohydrate-active enzymes is difficult due to low sequence identity. However, similar enzymes often share a few short motifs, e.g., around the active site, even when the overall sequences are very different. To exploit this notion for functional prediction of carbohydrate-active enzymes, we developed a simple algorithm, peptide pattern recognition (PPR), that can divide proteins into groups of sequences that share a set of short conserved sequences. When this method was used on 118 glycoside hydrolase 5 proteins with 9% average pairwise identity and representing four characterized enzymatic functions, 97% of the proteins were sorted into groups correlating with their enzymatic activity. Furthermore, we analyzed 8,138 glycoside hydrolase 13 proteins including 204 experimentally characterized enzymes with 28 different functions. There was a 91% correlation between group and enzyme activity. These results indicate that the function of carbohydrate-active enzymes can be predicted with high precision by finding short, conserved motifs in their sequences. The glycoside hydrolase 61 family is important for fungal biomass conversion, but only a few proteins of this family have been functionally characterized. Interestingly, PPR divided 743 glycoside hydrolase 61 proteins into 16 subfamilies useful for targeted investigation of the function of these proteins and pinpointed three conserved motifs with putative importance for enzyme activity. Furthermore, the conserved sequences were useful for cloning of new, subfamily-specific glycoside hydrolase 61 proteins from 14 fungi. In conclusion, identification of conserved sequence motifs is a new approach to sequence analysis that can predict carbohydrate-active enzyme functions with high precision.