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.

CRISPR/Cas-based genetic engineering has revolutionised molecular biology in both eukaryotes and prokaryotes. Several tools dedicated to the genomic transformation of the Clostridium genus of Gram-positive bacteria have been described in the literature; however, the integration of large DNA fragments still remains relatively limited. In this study, a CRISPR/Cas9 genome editing tool using a two-plasmid strategy was developed for the solventogenic strain Clostridium acetobutylicum ATCC 824. Codon-optimised cas9 from Streptococcus pyogenes was placed under the control of an anhydrotetracycline-inducible promoter on one plasmid, while the gRNA expression cassettes and editing templates were located on a second plasmid. Through the sequential introduction of these vectors into the cell, we achieved highly accurate genome modifications, including nucleotide substitution, gene deletion and cassette insertion up to 3.6kb. To demonstrate its potential, this genome editing tool was used to generate a marker-free mutant of ATCC 824 that produced an isopropanol-butanol-ethanol mixture. Whole-genome sequencing confirmed that no off-target modifications were present in the mutants. Such a tool is a prerequisite for efficient metabolic engineering in this solventogenic strain and provides an alternative editing strategy that might be applicable to other Clostridium strains.

Fuel oxygenates, mainly methyl tert-butyl ether (MTBE) but also ethyl tert-butyl ether (ETBE), are added to gasoline in replacement of lead tetraethyl to enhance its octane index. Their addition also improves the combustion efficiency and therefore decreases the emission of pollutants (CO and hydrocarbons). On the other hand, MTBE, being highly soluble in water and recalcitrant to biodegradation, is a major pollutant of water in aquifers contaminated by MTBE-supplemented gasoline during accidental release. MTBE was shown to be degraded through cometabolic oxidation or to be used as a carbon and energy source by a few microorganisms. We have summarized the present state of knowledge about the microorganisms involved in MTBE degradation and the MTBE catabolic pathways. The role of the different enzymes is discussed as well as the rare and recent data concerning the genes encoding the enzymes involved in the MTBE pathway. The phylogeny of the microorganisms isolated for their capacity to grow on MTBE is also described.

Here an overview of all pathways that integrate chemical and biological catalysis is presented. We emphasize the factors to be considered in order to understand catalytic synergy.