Challenging the workhorse: Comparative analysis of eukaryotic micro-organisms for expressing monoclonal antibodies.

For commercial protein therapeutics, Chinese hamster ovary (CHO) cells have an established history of safety, proven capability to express a wide range of therapeutic proteins and high volumetric productivities. Expanding global markets for therapeutic proteins and increasing concerns for broadened access of these medicines has catalyzed consideration of alternative approaches to this platform. Reaching these objectives likely will require an order of magnitude increase in volumetric productivity and a corresponding reduction in the costs of manufacture. For CHO-based manufacturing, achieving this combination of targeted improvements presents challenges. Based on a holistic analysis, the choice of host cells was identified as the single most influential factor for both increasing productivity and decreasing costs. Here we evaluated eight wild-type eukaryotic micro-organisms with prior histories of recombinant protein expression. The evaluation focused on assessing the potential of each host, and their corresponding phyla, with respect to key attributes relevant for manufacturing, namely (a) growth rates in industry-relevant media, (b) adaptability to modern techniques for genome editing, and (c) initial characterization of product quality. These characterizations showed that multiple organisms may be suitable for production with appropriate engineering and development and highlighted that yeast in general present advantages for rapid genome engineering and development cycles.

Directed evolution of a maltogenic alpha-amylase from Bacillus sp. TS-25.

Directed evolution coupled with a high-throughput robotic screen was employed to broaden the industrial use of the maltogenic alpha-amylase Novamyl from Bacillus sp. TS-25. Wild-type Novamyl is currently used in the baking industry as an anti-staling agent in breads baked at neutral or near neutral pH. However, the enzyme is rapidly inactivated during the baking process of bread made with low pH recipes and Novamyl thus has very limited beneficial effect for this particular application. In an effort to improve the performance of Novamyl for low pH bread applications such as sourdough and rye, two error-prone PCR libraries were generated, expressed in Bacillus subtilis and screened for variants with improved thermal stability and activity under low pH conditions. Variants exhibiting improved performance were iteratively recombined using DNA shuffling to create two generations of libraries. Relative to wild-type Novamyl, a number of the resulting variants exhibited more than 10 degrees C increase in thermal stability at pH 4.5, one of which demonstrated substantial anti-staling properties in low pH breads.

Evaluation of minimal Trichoderma reesei cellulase mixtures on differently pretreated Barley straw substrates.

The commercial cellulase product Celluclast 1.5, derived from Trichoderma reesei (Novozymes A/S, Bagsvaerd, Denmark), is widely employed for hydrolysis of lignocellulosic biomass feedstocks. This enzyme preparation contains a broad spectrum of cellulolytic enzyme activities, most notably cellobiohydrolases (CBHs) and endo-1,4-beta-glucanases (EGs). Since the original T. reesei strain was isolated from decaying canvas, the T. reesei CBH and EG activities might be present in suboptimal ratios for hydrolysis of pretreated lignocellulosic substrates. We employed statistically designed combinations of the four main activities of Celluclast 1.5, CBHI, CBHII, EGI, and EGII, to identify the optimal glucose-releasing combination of these four enzymes to degrade barley straw substrates subjected to three different pretreatments. The data signified that EGII activity is not required for efficient lignocellulose hydrolysis when addition of this activity occurs at the expense of the remaining three activities. The optimal ratios of the remaining three enzymes were similar for the two pretreated barley samples that had been subjeced to different hot water pretreatments, but the relative levels of EGI and CBHII activities required in the enzyme mixture for optimal hydrolysis of the acid-impregnated, steam-exploded barley straw substrate were somewhat different from those required for the other two substrates. The optimal ratios of the cellulolytic activities in all cases differed from that of the cellulases secreted by T. reesei. Hence, the data indicate the feasibility of designing minimal enzyme mixtures for pretreated lignocellulosic biomass by careful combination of monocomponent enzymes. This strategy can promote both a more efficient enzymatic hydrolysis of (ligno)cellulose and a more rational utilization of enzymes.

A proteomics strategy to discover beta-glucosidases from Aspergillus fumigatus with two-dimensional page in-gel activity assay and tandem mass spectrometry.

Economically competitive production of ethanol from lignocellulosic biomass by enzymatic hydrolysis and fermentation is currently limited, in part, by the relatively high cost and low efficiency of the enzymes required to hydrolyze cellulose to fermentable sugars. Discovery of novel cellulases with greater activity could be a critical step in overcoming this cost barrier. beta-Glucosidase catalyzes the final step in conversion of glucose polymers to glucose. Despite the importance, only a few beta-glucosidases are commercially available, and more efficient ones are clearly needed. We developed a proteomics strategy aiming to discover beta-glucosidases present in the secreted proteome of the cellulose-degrading fungus Aspergillus fumigatus. With the use of partial or complete protein denaturing conditions, the secretory proteome was fractionated in a 2DGE format and beta-glucosidase activity was detected in the gel after infusion with a substrate analogue that fluoresces upon hydrolysis. Fluorescing spots were subjected to tryptic-digestion, and identification as beta-glucosidases was confirmed by tandem mass spectrometry. Two novel beta-glucosidases of A. fumigatus were identified by this in situ activity staining method, and the gene coding for a novel beta-glucosidase ( EAL88289 ) was cloned and heterologously expressed. The expressed beta-glucosidase showed far superior heat stability to the previously characterized beta-glucosidases of Aspergillus niger and Aspergillus oryzae. Improved heat stability is important for development of the next generation of saccharifying enzymes capable of performing fast cellulose hydrolysis reactions at elevated temperatures, thereby lowering the cost of bioethanol production. The in situ activity staining approach described here would be a useful tool for cataloguing and assessing the efficiency of beta-glucosidases in a high throughput fashion.

Efficiency of new fungal cellulase systems in boosting enzymatic degradation of barley straw lignocellulose.

This study examined the cellulytic effects on steam-pretreated barley straw of cellulose-degrading enzyme systems from the five thermophilic fungi Chaetomium thermophilum, Thielavia terrestris, Thermoascus aurantiacus, Corynascus thermophilus, and Myceliophthora thermophila and from the mesophile Penicillum funiculosum. The catalytic glucose release was compared after treatments with each of the crude enzyme systems when added to a benchmark blend of a commercial cellulase product, Celluclast, derived from Trichoderma reesei and a beta-glucosidase, Novozym 188, from Aspergillus niger. The enzymatic treatments were evaluated in an experimental design template comprising a span of pH (3.5-6.5) and temperature (35-65 degrees C) reaction combinations. The addition to Celluclast + Novozym 188 of low dosages of the crude enzyme systems, corresponding to 10 wt % of the total enzyme protein load, increased the catalytic glucose yields significantly as compared to those obtained with the benchmark Celluclast + Novozyme 188 blend. A comparison of glucose yields obtained on steam-pretreated barley straw and microcrystalline cellulose, Avicel, indicated that the yield improvements were mainly due to the presence of highly active endoglucanase activity/activities in the experimental enzyme preparations. The data demonstrated the feasibility of boosting the widely studied T. reeseicellulase enzyme system with additional enzymatic activity to achieve faster lignocellulose degradation. We conclude that this supplementation strategy appears feasible as a first step in identifying truly promising fungal enzyme sources for fast development of improved, commercially viable, enzyme preparations for lignocellulose degradation.

Directed evolution of industrial enzymes: an update.

The use of enzymes in industrial processes can often eliminate the use of high temperatures, organic solvents and extremes of pH, while at the same time offering increased reaction specificity, product purity and reduced environmental impact. The growing use of industrial enzymes is dependent on constant innovation to improve performance and reduce cost. This innovation is driven by a rapidly increasing database of natural enzyme diversity, recombinant DNA and fermentation technologies that allow this diversity to be produced at low cost, and protein modification tools that enable enzymes to be tuned to fit into the industrial marketplace.

Spectroscopic characterization of the Leu513His variant of fungal laccase: effect of increased axial ligand interaction on the geometric and electronic structure of the type 1 Cu site.

A variety of spectroscopic techniques, combined with density functional calculations, are used to describe the electronic structure of the Leu513His variant of the type 1 Cu site in Myceliophthora thermophila laccase. This mutation changes the type 1 Cu from a blue to a green site. Electron paramagnetic resonance (EPR), optical absorption, circular dichroism, and magnetic circular dichroism (MCD) spectroscopies reveal that, relative to the trigonal planar blue type 1 Cu site in wild-type fungal laccase, the covalency and the ligand field strength at the Leu513His green type 1 Cu center decrease. Additionally, there is a significant reorientation of the d(x)()()2(-)(y)()()2( )singly occupied MO, such that the overlap with the Cys sulfur valence orbital changes from pi to sigma. A density functional study in which internal coordinates are systematically altered reveals that these changes are due to the increased strength of the axial ligand (none to His), leading to a tetragonal distortion and elongation of the equatorial Cu-ligand bonds. These calculations provide insight into the experimental differences in the EPR parameters, charge-transfer absorption spectrum, and ligand-field MCD spectrum between the axial-His variant and blue Cu centers (plastocyanin and the type 1 site in fungal laccase). There are also significant differences between the green site in the Leu513His variant and other naturally occurring, green type 1 Cu sites such as in nitrite reductase, which have short axial Cu-S(Met) bonds. The large difference in EPR parameters between these green type 1 sites derives from a change in ligand field excitation energies observed by MCD, which reflects a decrease in ligand field strength. This is associated with different steric interactions of a His vs an axial Met ligand in a tetragonally distorted type 1 site. Changes in the electronic structure of the Cu site correlate with the difference in reactivity of the green His variant relative to blue wild-type fungal laccase.