Engineering chimeric thermostable GH7 cellobiohydrolases in Saccharomyces cerevisiae.

We report here the effect of adding different types of carbohydrate-binding modules (CBM) to a single-module GH7 family cellobiohydrolase Cel7A from a thermophilic fungus Talaromyces emersonii (TeCel7A). Both bacterial and fungal CBMs derived from families 1, 2 and 3, all reported to bind to crystalline cellulose, were used. Chimeric cellobiohydrolases with an additional S-S bridge in the catalytic module of TeCel7A were also made. All the fusion proteins were secreted in active form and in good yields by Saccharomyces cerevisiae. The purified chimeric enzymes bound to cellulose clearly better than the catalytic module alone and demonstrated high thermal stability, having unfolding temperatures (T m) ranging from 72 °C to 77 °C. The highest activity enhancement on microcrystalline cellulose could be gained by a fusion with a bacterial CBM3 derived from Clostridium thermocellum cellulosomal-scaffolding protein CipA. The two CBM3 fusion enzymes tested were more active than the reference enzyme Trichoderma reesei Cel7A both at moderate (45 °C and 55 °C) and at high temperatures (60 °C and 65 °C), the hydrolysis yields being two- to three-fold better at 60 °C, and six- to seven-fold better at 65 °C. The best enzyme variant was also tested on a lignocellulosic feedstock hydrolysis, which demonstrated its potency in biomass hydrolysis even at 70 °C.

Improving the thermostability and activity of Melanocarpus albomyces cellobiohydrolase Cel7B.

Two different types of approach were taken to improve the hydrolytic activity towards crystalline cellulose at elevated temperatures of Melanocarpus albomyces Cel7B (Ma Cel7B), a single-module GH-7 family cellobiohydrolase. Structure-guided protein engineering was used to introduce an additional tenth disulphide bridge to the Ma Cel7B catalytic module. In addition, a fusion protein was constructed by linking a cellulose-binding module (CBM) and a linker from the Trichoderma reesei Cel7A to the C terminus of Ma Cel7B. Both approaches proved successful. The disulphide bridge mutation G4C/M70C located near the N terminus, close to the entrance of the active site tunnel of Ma Cel7B, led to improved thermostability (DeltaT (m) = 2.5 degrees C). By adding the earlier found thermostability-increasing mutation S290T (DeltaT (m) = 1.5 degrees C) together with the disulphide bridge mutation, the unfolding temperature was increased by 4 degrees C (mutant G4C/M70C/S290T) compared to that of the wild-type enzyme, thus showing an additive effect on thermostability. Both disulphide mutants had increased activity towards microcrystalline cellulose (Avicel) at 75 degrees C, apparently solely because of their improved thermostability. The addition of a CBM also improved the thermostability (DeltaT (m) = 2.5 degrees C) and caused a clear (sevenfold) increase in the hydrolysis activity of Ma Cel7B towards Avicel at 70 degrees C.

Identification in the yeast Pichia stipitis of the first L-rhamnose-1-dehydrogenase gene.

There are two distinctly different pathways for the catabolism of l-rhamnose in microorganisms. One pathway with phosphorylated intermediates was described in bacteria; here the enzymes and the corresponding gene sequences are known. The other pathway has no phosphorylated intermediates and has only been described in eukaryotic microorganisms. For this pathway, the enzyme activities have been described but not the corresponding gene sequences. The first enzyme in this catabolic pathway is the NAD-utilizing L-rhamnose 1-dehydrogenase. The enzyme was purified from the yeast Pichia stipitis, and the mass of its tryptic peptides was determined using MALDI-TOF MS. This enabled the identification of the corresponding gene, RHA1. It codes for a protein with 258 amino acids belonging to the protein family of short-chain alcohol dehydrogenases. The ORF was expressed in Saccharomyces cerevisiae. As the gene contained a CUG codon that codes for serine in P. stipitis but for leucine in S. cerevisiae, this codon has changed so that the same amino acid was expressed in S. cerevisiae. The heterologous protein showed the highest activity and affinity with L-rhamnose and a lower activity and affinity with L-mannose and L-lyxose. The enzyme was specific for NAD. A northern blot analysis revealed that transcription in P. stipitis is induced during growth on L-rhamnose but not on other carbon sources.

Cloning, expression, and characterization of novel thermostable family 7 cellobiohydrolases.

As part of the effort to find better cellulases for bioethanol production processes, we were looking for novel GH-7 family cellobiohydrolases, which would be particularly active on insoluble polymeric substrates and participate in the rate-limiting step in the hydrolysis of cellulose. The enzymatic properties were studied and are reported here for family 7 cellobiohydrolases from the thermophilic fungi Acremonium thermophilum, Thermoascus aurantiacus, and Chaetomium thermophilum. The Trichoderma reesei Cel7A enzyme was used as a reference in the experiments. As the native T. aurantiacus Cel7A has no carbohydrate-binding module (CBM), recombinant proteins having the CBM from either the C. thermophilum Cel7A or the T. reesei Cel7A were also constructed. All these novel acidic cellobiohydrolases were more thermostable (by 4-10 degrees C) and more active (two- to fourfold) in hydrolysis of microcrystalline cellulose (Avicel) at 45 degrees C than T. reesei Cel7A. The C. thermophilum Cel7A showed the highest specific activity and temperature optimum when measured on soluble substrates. The most effective enzyme for Avicel hydrolysis at 70 degrees C, however, was the 2-module version of the T. aurantiacus Cel7A, which was also relatively weakly inhibited by cellobiose. These results are discussed from the structural point of view based on the three-dimensional homology models of these enzymes.

High level secretion of cellobiohydrolases by Saccharomyces cerevisiae.

BACKGROUND: The main technological impediment to widespread utilization of lignocellulose for the production of fuels and chemicals is the lack of low-cost technologies to overcome its recalcitrance. Organisms that hydrolyze lignocellulose and produce a valuable product such as ethanol at a high rate and titer could significantly reduce the costs of biomass conversion technologies, and will allow separate conversion steps to be combined in a consolidated bioprocess (CBP). Development of Saccharomyces cerevisiae for CBP requires the high level secretion of cellulases, particularly cellobiohydrolases. RESULTS: We expressed various cellobiohydrolases to identify enzymes that were efficiently secreted by S. cerevisiae. For enhanced cellulose hydrolysis, we engineered bimodular derivatives of a well secreted enzyme that naturally lacks the carbohydrate-binding module, and constructed strains expressing combinations of cbh1 and cbh2 genes. Though there was significant variability in the enzyme levels produced, up to approximately 0.3 g/L CBH1 and approximately 1 g/L CBH2 could be produced in high cell density fermentations. Furthermore, we could show activation of the unfolded protein response as a result of cellobiohydrolase production. Finally, we report fermentation of microcrystalline cellulose (Avicel™) to ethanol by CBH-producing S. cerevisiae strains with the addition of beta-glucosidase. CONCLUSIONS: Gene or protein specific features and compatibility with the host are important for efficient cellobiohydrolase secretion in yeast. The present work demonstrated that production of both CBH1 and CBH2 could be improved to levels where the barrier to CBH sufficiency in the hydrolysis of cellulose was overcome.

Expression of Talaromyces emersonii cellobiohydrolase Cel7A in Saccharomyces cerevisiae and rational mutagenesis to improve its thermostability and activity.

We report here a successful expression of a single-module GH-7 family cellobiohydrolase Cel7A from a thermophilic fungus Talaromyces emersonii (Te Cel7A) in Saccharomyces cerevisiae. The heterologous expression system allowed structure-guided protein engineering to improve the thermostability and activity of Te Cel7A. Altogether six different mutants aimed at introducing additional disulphide bridges to the catalytic module of Te Cel7A were designed. These included addition of five individual S-S bridges in or between the loops extending from the beta-sandwich fold, and located either near the active site tunnel or forming the tunnel in Te Cel7A. A triple mutant containing the three best S-S mutations was also engineered. Three out of five single S-S mutants all had clearly improved thermostability which was also reflected as improved Avicel hydrolysis efficiency at 75 degrees C. The best mutant was the triple mutant whose unfolding temperature was improved by 9 degrees C leading to efficient microcrystalline cellulose hydrolysis at 80 degrees C. All the additional S-S bonds contributed mainly to the thermostability of the Te Cel7A, but one of the mutants (N54C/P191C) also showed, somewhat surprisingly, improved activity even at room temperature.