Selection of recombinant yeast that produces enzyme cocktails for application in consolidated bioprocessing

Abstract

Consolidated Bioprocessing (CBP) is an emerging technology for biomass processing, where the production of hydrolytic enzymes, biomass hydrolysis and sugar fermentation occur simultaneously in a single bioreactor through the application of microorganisms that secrete hydrolytic enzymes, eliminating the need for commercial enzymes cocktails. In general, the application of process engineering in CBP is little studied in the literature, where numerous works are found focused on the development of the recombinant strain. Considering the excellent performance in previous work of the group using the yeast Saccharomyces cerevisiae AC14, producer of 7 hydrolytic enzymes, in this project the CBP will be explored with a new perspective, where the production of the enzyme cocktail will be the protagonist of the process, so that the cocktail could be part of the biorefinery product portfolio or used in the hydrolysis of biomass to produce products of great industrial interest. More recently, two new yeasts have been developed from the AC14 strain: Cellusec 1® and Cellusec 3.1®. These strains overexpress the same 7 hydrolytic enzymes and are also resistant to inhibitors formed during biomass pretreatment. Considering that artificially introduced genetic modifications can reduce the robustness of S. cerevisiae, the present project aims to characterize the enzyme cocktails produced and to select the most suitable enzyme cocktail and yeast to be applied in the development of a scalable technology for CBP from bagasse and sugarcane straw, with the aim of producing ethanol and enzyme cocktail. Firstly, the enzymes produced and secreted by the three yeasts will be characterized in terms of their properties, stability under process conditions and catalytic capacity, through electrophoresis and measurement of the enzymatic activities of cellulase, xylanase, ²-xylosidase and ²-glucosidase, carried out at the optimum temperature for each enzyme and at 35ºC (temperature generally used in CBP). Next, the efficiency of enzyme cocktails produced in the hydrolysis of hydrothermally pretreated bagasse and sugarcane straw will be evaluated, and the yeasts AC14, Cellusec 1® and Cellusec 3.1® will be applied to bagasse and straw BPC. The aim is to select the best enzyme cocktail for biomass hydrolysis (at the optimal pH and temperature for hydrolysis) and the most suitable strain for BPC of these lignocellulosic materials. Finally, the enzyme cocktail and the selected strains will be scaled up in benchtop bioreactors specially designed by the group for each specificity of the enzymatic hydrolysis and CBP processes.