| Bioethanol synthesis from lignocellulosic substrates begins with the de-lignification and separation of the complex plant cell wall to the constitutent fermentable elements, primarily, cellulose (~50%) and hemicellulose (~30%). This occurs in a series of treatment steps prior to fermentation, including a cost-intensive enzymatic hydrolysis stage involving a cocktail of glycosylhydrolases and other accessory enzymes that hydrolyze the polysaccharides to simple, fermentable sugars. The goal of this research was to improve the efficiency and cost-effectiveness of enzymatic hydrolysis in pretreatment. The first strategy was to increase the secreted level of two model thermostable fungal hydrolases: a Thermoascus aurantiacus endoglucanase (Eg1) and a Thermomyces lanuginosus endoxylanase (XynA) from the host Pichia pastoris by genetic and growth strategies. Our results suggested that while codon optimization of the gene sequences showed no improvement, increasing the gene copy number and modulating culture conditions were successful in improving the enzyme level. The second strategy was to improve the specific activity of these hydrolases by using a rational mutagenesis approach. We developed two variants of the Eg1 enzyme: Eg1F16S and Eg1Y95F, with 1.7- and 4.0-fold improvements, respectively, in catalytic efficiency relative to wild-type Eg1. The double mutant Eg1F16SY95F showed a 60% decrease in catalytic efficiency. In contrast to the single mutation Eg1 variants, the two XynA variants: XynAW18Y and XynAN71Y, retained only 10% and 60% of the wild-type activity, respectively. This indicated that both positions were critical to maintaining the functional integrity of the enzyme. Finally, we developed a set of five trimeric xylanosomes and three trimeric cellulosomes for the combined saccharification and fermentation of hemicellulose and cellulose in Saccharomyces cerevisiae. The xylanosomes were evaluated for enzyme-enzyme and enzyme-substrate synergy, and for the binding module choice and position within the scaffoldin. The xylanosome carrying the Thermotoga maritima xylan binding module at a position closest to the endoxylanase showed a 3.3-fold improvement in hydrolysis over free enzymes. Similarly, the influence of enzyme position on cellulose hydrolysis was evaluated using the trimeric cellulosomes. The strategies developed in this research will be applicable to other pretreatment enzymes of interest for the enhanced hydrolysis of complex plant substrates in yeast. |
