Construction of cellulosic ethanol production process via Bacillus-yeast co-culturing strategy

• Main Authors: Cheng-Yu Ho, 何政育
• Other Authors: 黃介辰
• Format: Others
• Language: en_US
• Published: 2012
• Online Access: http://ndltd.ncl.edu.tw/handle/94957948175757819022

博士 === 國立中興大學 === 生命科學系所 === 100 === The kefir yeasts that produce ethanol and higher aromatic alcohols not only create kefir’s distinctive characteristics but could also serve as a good cell-based bioethanol production and biorefinery platform. This Kluyveromyces marxianus KY3 strain possesses a broad spectrum of substrate utilization, has a high potential for bioethanol production at elevated temperatures and suitable for expressing heterologous genes. We developed one synthetic biology techniques to transform genes into the host genome, named Promoter-based Gene Assembly and Simultaneous Overexpression 〔PGASO〕, which employed overlapping oligonucleotides for recombinatorial assembly of gene cassettes with individual unique promoters. As an example of application, PGASO was used to engineer yeast K. marxianus KY3. Our data showed high transformation efficiency and accuracy because ~63% of the transformants were found to carry the correct five-gene cassette assembly. And K. marxianus KY3-NpaBGS, which carries a β-glucosidase (NpaBGS) gene from rumen fungus. The two transgenic strains were employed to process ethanol production. “Designer cellulosomes” is a concept for making an artificial cellulosome that can be proposed as a tool for regulating the cellulosomal enzyme ratio to apply on different lignocellulose degradation. Two types of “designer operon” were expressed in Bacillus subtilis 168 via biomimetic approach according to a proteome-wide analysis of Clostridium thermocellum ATCC27405, which induced by avicel and cellobiose respectively. Eight celulosomal genes including one scaffolding protein gene (cipA), one cell-surface anchor gene (sdbA), two exoglucosidase genes (celK and celS), two endoglucanase genes (celA and cel R), and two xylanase genes (xynC and xynZ) of C. thermocellum were cloned and co-expressed on the polycistronic operons in desire order via an Ordered Gene Assembly in B. subtilis method (OGAB). A novel dual-microbe Bacillus/yeast co-culture system is developed for cellulosic bioethanol production. Two engineered yeasts, KY3-NpaBGS and KR5, which possess secretive cellulolytic enzymes, were used to co-cultivate with the B. subtilis type I strain for improving the cellulose digestion and fermentation efficiency. All Bacillus/yeast co-culture systems could achieve the cellulose saccharification and ethanol conversion simultaneously better than KR5 alone. In our result, the co-culturing of KY3-NpaBGS with Bacillus Type I appeared 6 times, 34% and 2.5 times ethanol production increasing than KY3+TypeI, KR5+ Type I and KY3-NpaBGS+host when utilized β-glucan as carbon source. Our results suggest that the dual-microbe Bacillus/yeast co-culturing system could leverage the advantages from both microbes and have a great potential for integrating into consolidated bioprocessing system.