Heterologous expression of cellulase genes from xanthomonas oryzae in saccharomyces cerevisiae for a one-step production of ethanol from rice straw

Name: Poravee Santiarphon

LCSH: Cellulase

Classification :.DDC: 572.756

Abstract: Lignocellulosic ethanol is the world next generation energy supply, though the entire process requires several steps and remains challenging. Heterologous expression of biomass hydrolyzing enzymes, cellulases, in yeast Saccharomyces cerevisiae, a mainstream microorganism in bioethanol industry, would reduce cost and time of bioethanol production significantly. Of all cellulase producing microorganisms, Xanthomonas oryzae pv. oryzae (Xoo), a rice pathogen causing blight leaf disease, might possess cellulases effective against rice straw, a major agricultural biomass in Thailand. Analyses of previous reports and bioinformatics of Xoo genome revealed that at least 12 cellulase genes existed in Xoo. In this study, we cloned 8 putative cellulase genes from Xoo. Of these genes, 3 endoglucanases (egl1, celB, and engxca) and a β-glucosidase (bglX1) were selected for further heterologous expression in yeast. Two approaches were used in our study. First, a direct in vivo homologous recombination of yeast genome with cellulase gene cassettes (egl1, and bglX1); however, we failed to score transgenic yeasts harboring cellulase genes due to an ineffective screening method. The second approach was to generate recombinant plasmid vectors via in vivo homologous recombination in yeast. These vectors contained suc2 promoter and suc2 signal sequences required for expression and secretion in which the suc2 promoter could be induced by sucrose and repressed by glucose. Cellulase genes such as celB, engxca, and bglX1 were successfully cloned and inserted downstream of suc2 promoter. Unfortunately, cellulase activity assays of the supernatant of the yeast cell culture or yeast cell extracts revealed that both recombinant endoglucanases and β-glucosidase enzymes had slight or no activity even though the presence of some recombinant proteins was verified by SDS-PAGE as a distinct band on a gel. Taken together, we conclude that most cellulases from Xoo require several enzymes clustered together to work as a modular protein systems or a cellulosome. Despite the fact that we have not been able to engineer yeast to be able to hydrolyze cellulose using rice straw as a sole substrate yet, we shed the light on the novel cellulases from a rice deadly pathogenic strain which might be applied to the future approach for a one-step production of ethanol from rice straw.

Burapha University. Library

Address: CHONBURI

Email: buulibrary@buu.ac.th

Name: Salil Chanroj

Role: Principal advisor

Created: 2020

Modified: 2021-06-18

Issued: 2021-06-18

วิทยานิพนธ์/Thesis

application/pdf

CallNumber: Th 572.756 Po832H

eng

DegreeName: Master of Science

Level: Master's Degree

Descipline: Biological Science

Grantor: Burapha University

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