There is arguably no other industrial enzyme application that poses a greater challenge to the enzyme producer than supplying cost-effective enzymes for biomass utilization. The high enzyme loading required, combined with the low value of the final product, in the form of ethanol, requires not only that the enzymes be as efficient as possible, but that the cost of producing them be as low as possible. To this end, significant effort has been expended over the past 6 years to increase the productivity of the fungal strains used to pro­duce the enzymes, to reduce the cost of the enzyme fermentation process by reducing the cost of carbon and nitrogen sources for the fermentations, and to reduce the complexity of enzyme recovery and formulation.

Improving the host by classical mutagenesis is one way of developing a host strain with improved total protein production and improved activ­ities. This approach has a long and successful history. Montenecourt and Eveleigh [32] isolated RutC30, one of the best existing Trichoderma cellulase mutants, using a combination of ultraviolet irradiation and nitrosomethyl guanidine (NTG). Recently, Toyama, et al. [45] demonstrated a method to screen for increased cellulase production using growth through an overlay of cellulose substrate (Avicel) in Petri plates. In an effort to increase total cellulase productivity, a combination of these methods were utilized on the T. reesei strain currently used to produce Celluclast 1.5 L. Chemical muta­genesis was used to generate mutants that were screened using the method of Toyama [45] with minor changes. Briefly, mutagenized spores were sus­pended in an agar medium, poured into a plate and allowed to harden. The spore-containing layer was then covered with a top layer of agar contain­ing washed, acid pretreated corn stover (PCS) as the sole carbon source. Colonies growing through the PCS layer fastest were isolated and used in a secondary screening. In this, spores from selected fast-growing colonies were inoculated into shake flasks containing cellulase-inducing media. After 5 days of growth, broth samples were tested by robotic assay for produc­tion of reducing sugars from hydrolysis of PCS. Total protein assays were then conducted, and mutants expressing elevated cellulase and/or total pro­tein were then re-grown in 2-L fermentors. Broth from the fermentors was then analyzed again in PCS hydrolysis assays and for total protein. Some mu­tants were identified as having improved PCS hydrolysis and increased total protein secretion compared with the control. Top strains isolated in this man­ner showed significant increases in protein production and secreted cellulase activity.

Another method of improving a cellulase productivity is through increas­ing the expression of target proteins using genetic engineering. In many cases the total cost of supplying a heterologous mix of enzymes can be reduced by creating a single expression host expressing not only the native cellulases and hemicellulases, but expressing additional components, such as the BG and GH61 proteins, without negatively impacting the expression of the na­tive proteins. The introduction of multiple genes into a single host is no easy feat. A significant amount of work was done to identify strong promoters, to identify a number of selectable markers, and to develop a successful trans­formation technique that allows for co-transformation of multiple transgenes. These technological improvements have allowed us to rapidly and efficiently investigate the effect of introducing various enzymes into the T. reesei cellu — lase mix.

In addition to controlling gene expression transcriptionally, by utilizing promoters of different strengths, we have focused on enhancing individual protein yield by optimizing protein secretion. One example is the replacement of the A. oryzae BG signal sequence with a signal peptide from H. insolens Cel45A EG, which improved the BG secretion in T. reesei by two- to threefold relative to the unfused gene (Fig. 10).

As previously mentioned, several GH61 proteins result in a “boost” in PCS hydrolysis when supplemented to Celluclast 1.5 L. In addition, our stud­ies show that increased levels of в-glucosidase are required in our Tricho — derma host. Therefore, numerous co-transformations of T. reesei with various GH61s, A. oryzae в-glucosidase, and other genes of interest were carried out. Those transformants expressing both a GH61 and the в-glucosidase were then screened in PCS hydrolysis assays in order to identify the top strains in true performance assays. Those strains demonstrating the best perform-

ance in PCS hydrolysis were then fermented in 2-L bioreactors and retested in PCS hydrolysis assays. Eventually, a single strain was identified exhibiting im­proved hydrolysis from our original strains and high total protein production (Fig. 11).

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