Teolenn: an efficient and customizable workflow to design high-quality probes for microarray experiments.

Despite the development of new high-throughput sequencing techniques, microarrays are still attractive tools to study small genome organisms, thanks to sample multiplexing and high-feature densities. However, the oligonucleotide design remains a delicate step for most users. A vast array of software is available to deal with this problem, but each program is developed with its own strategy, which makes the choice of the best solution difficult. Here we describe Teolenn, a universal probe design workflow developed with a flexible and customizable module organization allowing fixed or variable length oligonucleotide generation. In addition, our software is able to supply quality scores for each of the designed probes. In order to assess the relevance of these scores, we performed a real hybridization using a tiling array designed against the Trichoderma reesei fungus genome. We show that our scoring pipeline correlates with signal quality for 97.2% of all the designed probes, allowing for a posteriori comparisons between quality scores and signal intensities. This result is useful in discarding any bad scoring probes during the design step in order to get high-quality microarrays. Teolenn is available at http://transcriptome.ens.fr/teolenn/.

Tracking the roots of cellulase hyperproduction by the fungus Trichoderma reesei using massively parallel DNA sequencing.

Trichoderma reesei (teleomorph Hypocrea jecorina) is the main industrial source of cellulases and hemicellulases harnessed for the hydrolysis of biomass to simple sugars, which can then be converted to biofuels such as ethanol and other chemicals. The highly productive strains in use today were generated by classical mutagenesis. To learn how cellulase production was improved by these techniques, we performed massively parallel sequencing to identify mutations in the genomes of two hyperproducing strains (NG14, and its direct improved descendant, RUT C30). We detected a surprisingly high number of mutagenic events: 223 single nucleotides variants, 15 small deletions or insertions, and 18 larger deletions, leading to the loss of more than 100 kb of genomic DNA. From these events, we report previously undocumented non-synonymous mutations in 43 genes that are mainly involved in nuclear transport, mRNA stability, transcription, secretion/vacuolar targeting, and metabolism. This homogeneity of functional categories suggests that multiple changes are necessary to improve cellulase production and not simply a few clear-cut mutagenic events. Phenotype microarrays show that some of these mutations result in strong changes in the carbon assimilation pattern of the two mutants with respect to the wild-type strain QM6a. Our analysis provides genome-wide insights into the changes induced by classical mutagenesis in a filamentous fungus and suggests areas for the generation of enhanced T. reesei strains for industrial applications such as biofuel production.

Transcriptional profiling of cellulase and expansin-related genes in a hypercellulolytic Trichoderma reesei.

Expression kinetics of six cellulase and four expansin-related genes were studied in the hypercellulolytic Trichoderma reesei CL847 mutant in response to Solka Floc cellulose and soluble inducers. Real-time PCR showed a parallel increase of transcript levels for the cellulase genes cbh1/cel7a, egl1/cel7b, egl4/cel61a, the beta-glucosidase genes bgl1/cel3a, bgl2/cel1a, and the swo1 gene, encoding the cell-wall loosening protein swollenin. To evaluate a putative implication of three newly identified expansin/family 45 endoglucanase-like (EEL) proteins in lignocellulose degradation, their expression was also analysed. Only eel2 was found to be transcribed under the present conditions, and showed constitutive expression similar to the endoglucanase encoding cel5b gene.

Comparative kinetic analysis of two fungal beta-glucosidases.

BACKGROUND: The enzymatic hydrolysis of cellulose is still considered as one of the main limiting steps of the biological production of biofuels from lignocellulosic biomass. It is a complex multistep process, and various kinetic models have been proposed. The cellulase enzymatic cocktail secreted by Trichoderma reesei has been intensively investigated. beta-glucosidases are one of a number of cellulolytic enzymes, and catalyze the last step releasing glucose from the inhibitory cellobiose. beta-glucosidase (BGL1) is very poorly secreted by Trichoderma reesei strains, and complete hydrolysis of cellulose often requires supplementation with a commercial beta-glucosidase preparation such as that from Aspergillus niger (Novozymes SP188). Surprisingly, kinetic modeling of beta-glucosidases lacks reliable data, and the possible differences between native T. reesei and supplemented beta-glucosidases are not taken into consideration, possibly because of the difficulty of purifying BGL1. RESULTS: A comparative kinetic analysis of beta-glucosidase from Aspergillus niger and BGL1 from Trichoderma reesei, purified using a new and efficient fast protein liquid chromatography protocol, was performed. This purification is characterized by two major steps, including the adsorption of the major cellulases onto crystalline cellulose, and a final purification factor of 53. Quantitative analysis of the resulting beta-glucosidase fraction from T. reesei showed it to be 95% pure. Kinetic parameters were determined using cellobiose and a chromogenic artificial substrate. A new method allowing easy and rapid determination of the kinetic parameters was also developed. beta-Glucosidase SP188 (Km = 0.57 mM; Kp = 2.70 mM) has a lower specific activity than BGL1 (Km = 0.38 mM; Kp = 3.25 mM) and is also more sensitive to glucose inhibition. A Michaelis-Menten model integrating competitive inhibition by the product (glucose) has been validated and is able to predict the beta-glucosidase activity of both enzymes. CONCLUSIONS: This article provides a useful comparison between the activity of beta-glucosidases from two different fungi, and shows the importance of fully characterizing both enzymes. A Michaelis-Menten model was developed, including glucose inhibition and kinetic parameters, which were accurately determined and compared. This model can be further integrated into a cellulose hydrolysis model dissociating beta-glucosidase activity from that of other cellulases. It can also help to define the optimal enzymatic cocktails for new beta-glucosidase activities.

Use of Mycobacterium austroafricanum IFP 2012 in a MTBE-degrading bioreactor.

Mycobacterium austroafricanum IFP 2012 is able to slowly grow on methyl tert-butyl ether (MTBE), a fuel oxygenate widely used as a gasoline additive. The potential of M. austroafricanum IFP 2012 for aerobic MTBE degradation was investigated in the presence of a secondary carbon source, isopropanol. The strain was then tested for MTBE biodegradation at the laboratory-scale in a fixed-bed reactor using perlite as the matrix, and isopropanol was injected once a week to maintain M. austroafricanum IFP 2012 biomass inside the perlite bed. The biofilter was operated for 85 days at an influent flow rate of 20 ml/h by varying the MTBE concentration from 10 to 20 mg/l. The hydraulic retention time was fixed at 5 days. The removal of MTBE depended on the inlet MTBE concentration and a MTBE removal efficiency higher than 99% was obtained for MTBE concentrations up to 15 mg/l. A set of 16S rRNA gene primers specific for M. austroafricanum species was used to analyze the DNA extracted from the biofilter effluent in order to detect the presence of M. austroafricanum IFP 2012 and to estimate the effect of periodic injections of isopropanol on the release of the strain from the perlite bed. The results demonstrated that the injection of isopropanol served to maintain an active MTBE degrading biomass in the biofilter and that this system could be used to effectively treat MTBE contaminated groundwater.

n-Alkane assimilation and tert-butyl alcohol (TBA) oxidation capacity in Mycobacterium austroafricanum strains.

Mycobacterium austroafricanum IFP 2012, which grows on methyl tert-butyl ether (MTBE) and on tert-butyl alcohol (TBA), the main intermediate of MTBE degradation, also grows on a broad range of n-alkanes (C2 to C16). A single alkB gene copy, encoding a non-heme alkane monooxygenase, was partially amplified from the genome of this bacterium. Its expression was induced after growth on n-propane, n-hexane, n-hexadecane and on TBA but not after growth on LB. The capacity of other fast-growing mycobacteria to grow on n-alkanes (C1 to C16) and to degrade TBA after growth on n-alkanes was compared to that of M. austroafricanum IFP 2012. We studied M. austroafricanum IFP 2012 and IFP 2015 able to grow on MTBE, M. austroafricanum IFP 2173 able to grow on isooctane, Mycobacterium sp. IFP 2009 able to grow on ethyl tert-butyl ether (ETBE), M. vaccae JOB5 (M. austroaafricanum ATCC 29678) able to degrade MTBE and TBA and M. smegmatis mc2 155 with no known degradation capacity towards fuel oxygenates. The M. austroafricanum strains grew on a broad range of n-alkanes and three were able to degrade TBA after growth on propane, hexane and hexadecane. An alkB gene was partially amplified from the genome of all mycobacteria and a sequence comparison demonstrated a close relationship among the M. austroafricanum strains. This is the first report suggesting the involvement of an alkane hydroxylase in TBA oxidation, a key step during MTBE metabolism.

Isolation and characterization of a new Mycobacterium austroafricanum strain, IFP 2015, growing on MTBE.

A new Mycobacterium austroafricanum strain, IFP 2015, growing on methyl tert-butyl ether (MTBE) as a sole carbon source was isolated from an MTBE-degrading microcosm inoculated with drain water of an MTBE-supplemented gasoline storage tank. M. austroafricanum IFP 2015 was able to grow on tert-butyl formate, tert-butyl alcohol (TBA) and alpha-hydroxyisobutyrate. 2-Methyl-1,2-propanediol was identified as the TBA oxidation product in M. austroafricanum IFP 2015 and in the previously isolated M. austroafricanum IFP 2012. M. austroafricanum IFP 2015 also degraded ethyl tert-butyl ether more rapidly than M. austroafricanum IFP 2012. Specific primers designed to monitor the presence of M. austroafricanum strains could be used as molecular tools to detect similar strains in MTBE-contaminated environment.

Biodegradation of methyl tert-butyl ether and other fuel oxygenates by a new strain, Mycobacterium austroafricanum IFP 2012.

A strain that efficiently degraded methyl tert-butyl ether (MTBE) was obtained by initial selection on the recalcitrant compound tert-butyl alcohol (TBA). This strain, a gram-positive methylotrophic bacterium identified as Mycobacterium austroafricanum IFP 2012, was also able to degrade tert-amyl methyl ether and tert-amyl alcohol. Ethyl tert-butyl ether was weakly degraded. tert-Butyl formate and 2-hydroxy isobutyrate (HIBA), two intermediates in the MTBE catabolism pathway, were detected during growth on MTBE. A positive effect of Co2+ during growth of M. austroafricanum IFP 2012 on HIBA was demonstrated. The specific rate of MTBE degradation was 0.6 mmol/h/g (dry weight) of cells, and the biomass yield on MTBE was 0.44 g (dry weight) per g of MTBE. MTBE, TBA, and HIBA degradation activities were induced by MTBE and TBA, and TBA was a good inducer. Involvement of at least one monooxygenase during degradation of MTBE and TBA was shown by (i) the requirement for oxygen, (ii) the production of propylene epoxide from propylene by MTBE- or TBA- grown cells, and (iii) the inhibition of MTBE or TBA degradation and of propylene epoxide production by acetylene. No cytochrome P-450 was detected in MTBE- or TBA-grown cells. Similar protein profiles were obtained after sodium dodecyl sulfate-polyacrylamide gel electrophoresis of crude extracts from MTBE- and TBA-grown cells. Among the polypeptides induced by these substrates, two polypeptides (66 and 27 kDa) exhibited strong similarities with known oxidoreductases.