Expression of Heterologous Cellulases in Thermotoga sp. Strain RQ2.
The ability of Thermotoga spp. to degrade cellulose is limited due to a lack of exoglucanases. To address this deficiency, cellulase genes Csac_1076 (celA) and Csac_1078 (celB) from Caldicellulosiruptor saccharolyticus were cloned into T. sp. strain RQ2 for heterologous overexpression. Coding regions of Csac_1076 and Csac_1078 were fused to the signal peptide of TM1840 (amyA) and TM0070 (xynB), resulting in three chimeric enzymes, namely, TM1840-Csac_1078, TM0070-Csac_1078, and TM0070-Csac_1076, which were carried by Thermotoga-E. coli shuttle vectors pHX02, pHX04, and pHX07, respectively. All three recombinant enzymes were successfully expressed in E. coli DH5α and T. sp. strain RQ2, rendering the hosts with increased endo- and/or exoglucanase activities. In E. coli, the recombinant enzymes were mainly bound to the bacterial cells, whereas in T. sp. strain RQ2, about half of the enzyme activities were observed in the culture supernatants. However, the cellulase activities were lost in T. sp. strain RQ2 after three consecutive transfers. Nevertheless, this is the first time heterologous genes bigger than 1 kb (up to 5.3 kb in this study) have ever been expressed in Thermotoga, demonstrating the feasibility of using engineered Thermotoga spp. for efficient cellulose utilization.
Department of Biological Sciences, Bowling Green State University, Bowling Green, OH 43403, USA.
Hui
H
Xu
Department of Biological Sciences, Bowling Green State University, Bowling Green, OH 43403, USA.
Dongmei
D
Han
Department of Biological Sciences, Bowling Green State University, Bowling Green, OH 43403, USA.
Zhaohui
Z
Xu
D001426:Bacterial Proteins / Q000235:genetics*; D044602:Cellulases / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D006089:Gram-Negative Anaerobic Bacteria / Q000235:genetics*; D021382:Protein Sorting Signals / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics
D001426:Bacterial Proteins; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D044602:Cellulases