Background: Due to its capacity to produce large amounts of cellulases, Trichoderma reesei is increasingly been researched in various fields of white biotechnology, especially in biofuel production from lignocellulosic biomass. The commercial enzyme mixtures produced at industrial scales are not well characterized, and their proteinaceous components are poorly identified and quantified. The development of proteomic methods has made it possible to comprehensively overview the enzymes involved in lignocellulosic biomass degradation which are secreted under various environmental conditions.

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. biofuels ͉ biotechnology

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 Co 2؉ 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 TBAgrown 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.Methyl tert-butyl ether (MTBE) has been incorporated in reformulated gasoline at concentrations up to 15% (vol/vol) to replace lead tetraethyl in order to comply with the octane index and to reduce the polluting emissions in exhaust gases. Other oxygenates, such as ethyl tert-butyl ether (ETBE) and tert-amyl methyl ether (TAME) and their corresponding alcohols, tert-butyl alcohol (TBA) and tert-amyl alcohol (TAA), can play the same role regarding the octane index. MTBE is the dominant fuel oxygenate (38), with a worldwide production capacity of around 25 million tons. Because of its widespread use and the high frequency of underground tank leakage (35), this compound is now the second most commonly detected contaminant in urban groundwater in the United States (23, 42). In Europe, detectable levels of MTBE in rivers have also been reported (1).The persistence of MTBE in the environment can be ascribed, on the one hand, to its physicochemical properties (i.e., its low adsorption on organic matter and its high solubility in water) and, on the other hand, to its molecular structure (it has both an ether bond and high steric hindrance, which makes it recalcitrant to microbial degradation).Early reports based on microcosms having different origins mentioned the recalcitrance of MTBE and TAME (22) MTBE cometabolism was demonstrated by using propanegrown bacteria (35), an n-butane-grown fungus (17), camphorgrown Pseudomonas putida CAM (36), pentane-grown Pseudomonas aeruginosa (14), ETBE-grown Rhodococcus ruber IFP 2007 (...

BackgroundThe 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. β-glucosidases are one of a number of cellulolytic enzymes, and catalyze the last step releasing glucose from the inhibitory cellobiose. β-glucosidase (BGL1) is very poorly secreted by Trichoderma reesei strains, and complete hydrolysis of cellulose often requires supplementation with a commercial β-glucosidase preparation such as that from Aspergillus niger (Novozymes SP188). Surprisingly, kinetic modeling of β-glucosidases lacks reliable data, and the possible differences between native T. reesei and supplemented β-glucosidases are not taken into consideration, possibly because of the difficulty of purifying BGL1.ResultsA comparative kinetic analysis of β-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 β-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. β-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 β-glucosidase activity of both enzymes.ConclusionsThis article provides a useful comparison between the activity of β-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 β-glucosidase activity from that of other cellulases. It can also help to define the optimal enzymatic cocktails for new β-glucosidase activities.

No abstract

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.

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.

Mycobacterium austroafricanum IFP 2012 is a Gram-positive strain able to grow on methyl tert-butyl ether (MTBE) as a sole carbon and energy source. The effect of two downstream metabolites of MTBE, tert-butyl formate (TBF) and tert-butyl alcohol (TBA) on MTBE degradation was investigated using resting cells. The addition of low concentrations of TBF decreased the MTBE degradation rate by about 30%. In contrast, the addition of TBA did not have a significant effect on MTBE degradation rate, even at high concentrations; and it was also shown that TBA degradation occurred only once MTBE was exhausted. At neutral pH, TBF hydrolysis involved mainly an esterase-type activity regulated by the presence of TBA. The TBF degradation rate was about four times lower than the MTBE degradation rate. Furthermore, acetone was identified as an intermediate during TBA degradation. An acetone mono-oxygenase activity, inhibited by methimazole but not by acetylene, was suggested. It was different from the MTBE/TBA mono-oxygenase and, thus, acetone did not appear to compete with MTBE and TBA for the same enzyme. These new results show that the metabolic regulation of the early steps of MTBE degradation by M. austroafricanum IFP 2012 is complex, involving inhibition and competition phenomena.