Heterologous co-production of Thermobifida fusca Cel9A with other cellulases in Saccharomyces cerevisiae

• DOI: 10.1007/s00253-010-2618-z
• Publication Date: 2010-05-06T11:15:17Z
• Authors: Niel van Wyk; Riaan den Haan; Willem H. van Zyl
• Keywords: Endoglucanases; bacterial growth; Recombinant hosts; Thermobifida fusca; Phosphoric-acid swollen cellulose; S.cerevisiae; Cellulases; Amorphous cellulose; Trichoderma; cellulase; 0303 health sciences; article; Anionic exchange; Carboxy methylcellulose; bacterium; cel5a gene; Chromatography, Ion Exchange; Cellulose substrates; Recombinant Proteins; enzyme activity; Co-production; sugar; Cellobiohydrolases; chromatography; growth rate; bacterial gene; Cellulose chain; 570; cel6a gene; Trichoderma reesei; Carbohydrate sources; Carbohydrates; Soluble sugars; Saccharomyces cerevisiae; Moderately thermophilic; Fungal Proteins; Consolidated bioprocessing; 03 medical and health sciences; Bacterial Proteins; Actinomycetales; carboxymethylcellulose; Cellulose; Substrates; Cellulosic substrates; Coding genes; Recombinant strains; bacterial strain; Co-expression; Yeast; recombination; Glucose; cellobiose; Sugar (sucrose); gene expression; cel7a gene; Acids; Phosphoric acid

The processive endoglucanase Cel9A of the moderately thermophilic actinomycete Thermobifida fusca was functionally produced in Saccharomyces cerevisiae. Recombinant Cel9A displayed activity on both soluble (carboxymethylcellulose) and insoluble (Avicel) cellulose substrates confirming its processive endoglucanase activity. High-performance anionic exchange chromatography analyses of soluble sugars released from Avicel revealed a cellobiose/glucose ratio of 2.5 +/- 0.1. Growth by the recombinant strain on amorphous cellulose was possible due to the sufficient amount of glucose cleaved from the cellulose chain. This is the first confirmed report of S. cerevisiae growing on a cellulosic substrate as sole carbohydrate source while only expressing one recombinant gene. To improve the cellulolytic capability of S. cerevisiae and to investigate the level of synergy among cellulases produced by a recombinant host, the cel9A gene was co-expressed with four cellulase-coding genes of Trichoderma reesei: two endoglucanases cel5A (egII) and cel7B (egI), and two cellobiohydrolases cel6A (cbhII) and cel7A (cbhI). Synergy, especially between the Cel9A and the two cellobiohydrolases, resulted in a higher cellulolytic capability of the recombinant host.