Protein stability engineering by structure-guided chimeragenesis

Beta-glucosidase is one of the key enzymes that convert biomass glucans to a final product D-glucose. We identified a beta-glucosidase from Fusarium verticilloides (Fv3C) that displayed good performance on a panel of biomass substrates. However, when expressed in Trichoderma reesei, this molecule was proteolytically clipped. MS analysis, N-terminal sequencing, and X-ray crystallography showed that the protein got clipped within the C-terminal third domain resulting in a significant loss of performance. Attempts to fix the integrity of Fv3C locally, including site-specific mutations, loop replacement or deletions, linker or disulfide bridge insertions, and removal or introduction of N-linked glycosylation sites, were unsuccessful. Alternatively, we applied a “global” approach via interchanging regions between Fv3C and homologs. Replacing the C-terminal domain of Fv3C repaired the clip site, but surprisingly created another one in a loop upstream of the previous clip site. This loop is specific only to Fusarium beta-glucosidases and shortening this sequence to a single Gly residue or to a corresponding region from a homolog led to molecules which are produced intact by T. reesei. The three-way hybrid performed well and remained stable during saccharification assays.