Engineering of a functional thermostable kanamycin resistance marker for use in Moorella thermoacetica ATCC39073

Authors


Correspondence: Yutaka Nakashimada, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8530, Japan. Tel.: +81 82 424 4443; fax: +81 82 424 4443; e-mail: nyutaka@hiroshima-u.ac.jp

Abstract

A transformation system for Moorella thermoacetica ATCC39073 was developed using thermostable kanamycin resistant gene (kanR) derived from the plasmid pJH1 that Streptococcus faecalis harbored. When kanR with its native promoter was introduced into uracil auxotrophic mutant of Mthermoacetica ATCC39073 together with a gene to complement the uracil auxotrophy as a selection marker, it did not give kanamycin resistance due to poor transcription level of kanR. However, the use of glyceraldehyde-3-phosphate dehydrogenase promoter cloned from M. thermoacetica ATCC39073 significantly improved transcription level of kanR and resulted in the cell growth in the presence of more than 150 μg mL−1 kanamycin. It was also demonstrated that kanR with G3PD promoter can be used as a selection marker for transformation of wild-type strain of Mthermoacetica ATCC39073.

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