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Hui Xu-Dissertation 20150519.pdf (4.62 MB)

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Genetic Modification of Thermotoga to Degrade Cellulose

Xu, Hui
http://orcid.org/0000-0001-9707-5671
http://rave.ohiolink.edu/etdc/view?acc_num=bgsu1430913637

Abstract Details

2015, Doctor of Philosophy (Ph.D.), Bowling Green State University, Biological Sciences.
Thermotoga spp. can effectively utilize a variety of carbohydrates to generate hydrogen gas, making them great candidates for biofuel production. However, their ability to degrade cellulose, the most common and renewable organic material on Earth, is rather limited due to a lack of exo-glucanases. To address this deficiency, firstly technical preparations were done: the methylase M.TneDI was overexpressed in E. coli and could be used for in vitro methylation, which could enhance transformation efficiency of Thermotoga; a natural transformation protocol was optimized in T. sp. strain RQ2, which provided the convenience of genetic engineering of Thermotoga. Then cellulase genes Csac_1076 (celA) and Csac_1078 (celB) from Caldicellulosiruptor saccharolyticus DSM 8903 were cloned into T. sp. strain RQ2 for heterologous overexpression. Thermotoga-E. coli shuttle vectors pHX02, pHX04 and pHX07 carrying celA or celB were successfully constructed and naturally transformed into T. sp strain RQ2. Recombinant cellulases were successfully expressed and secreted outside hosts, and the resulting T. sp. strain RQ2 transformants demonstrated enhanced abilities of cellulose degradation (endo- and/or exo-glucanase activities). Even though the T. sp. strain RQ2 transformants are not stable as indicated by loss of enhanced cellulase activities after three consecutive transfers, this is still the first time heterologous genes larger than 1 kb (up to 5.3 kb) have been expressed in Thermotoga, and definitely a milestone of genetic engineering of Thermotoga to utilize cellulose.
Zhaohui Xu, PhD (Advisor)
R. Marshall Wilson, PhD (Other)
George Bullerjahn, PhD (Committee Member)
Robert McKay, PhD (Committee Member)
Scott Rogers, PhD (Committee Member)
119 p.
Biology
Thermotoga; Methylase; Restriction-modification system; Natural transformation; Cellulose; Cellulase

Recommended Citations

Citations

  • Xu, H. (2015). Genetic Modification of Thermotoga to Degrade Cellulose [Doctoral dissertation, Bowling Green State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=bgsu1430913637

    APA Style (7th edition)

  • Xu, Hui. Genetic Modification of Thermotoga to Degrade Cellulose. 2015. Bowling Green State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=bgsu1430913637.

    MLA Style (8th edition)

  • Xu, Hui. "Genetic Modification of Thermotoga to Degrade Cellulose." Doctoral dissertation, Bowling Green State University, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=bgsu1430913637

    Chicago Manual of Style (17th edition)

bgsu1430913637
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© 2015, all rights reserved.
This open access ETD is published by Bowling Green State University and OhioLINK.