Biology Ph.D. Dissertations

Genetic Modification of Thermotoga to Degrade Cellulose

Date of Award

2015

Document Type

Dissertation

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Biological Sciences

First Advisor

Zhaohui Xu (Advisor)

Second Advisor

R. Marshall Wilson (Other)

Third Advisor

George Bullerjahn (Committee Member)

Fourth Advisor

Robert McKay (Committee Member)

Fifth Advisor

Scott Rogers (Committee Member)

Abstract

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.

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