Design and construction of improved new vectors for Zymomonas mobilis recombinants

Biotechnology and Bioengineering - Tập 108 Số 7 - Trang 1616-1627 - 2011
Hongwei Dong1, Jie Bao2, Dewey D. Y. Ryu3, Jian‐Jiang Zhong4
1State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, China; fax: +86-21-64252250; telephone: +86-21-64251799
2Dewey D.Y. Ryu, Biochemical Engineering Program, University of California, One Shields Ave, Davis, California 95616; Jian-Jiang Zhong, Shanghai Jiao Tong University, School of Life Sciences and Biotechnology, Key Laboratory of Microbial Metabolism of Ministry of Education, Shanghai 200240, China; Jie Bao, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, China; fax: +1-530-752-3112.; fax: +86-21-64252250.; telephone: +1-530-752-8954; telephone: +86-21-34206968; telephone: +86-21-64251799
3Biochemical Engineering Program, University of California, One Shields Ave, Davis, California 95616; Jian-Jiang Zhong, Shanghai Jiao Tong University, School of Life Sciences and Biotechnology, Key Laboratory of Microbial Metabolism of Ministry of Education, Shanghai 200240, China; Jie Bao, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, China; fax: +1-530-752-3112; fax: +86-21-64252250.; telephone: +1-530-752-8954; telephone: +86-21-34206968; telephone: +86-21-64251799
4Dewey D.Y. Ryu, Biochemical Engineering Program, University of California, One Shields Ave, Davis, California 95616; Jie Bao, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, China; Shanghai Jiao Tong University, School of Life Sciences and Biotechnology, Key Laboratory of Microbial Metabolism of Ministry of Education, Shanghai 200240, China; fax: +1-530-752-3112.; fax: +86-21-64252250.; telephone: +1-530-752-8954; telephone: +86-21-34206968; telephone: +86-21-64251799

Tóm tắt

AbstractZymomonas mobilis is a very important gram‐negative bacterium having a potential application to simultaneous co‐production of biofuel and other high value‐added products through biorefinery process technology development. Up to now, pLOI193 has been used as the plasmid of choice for Z. mobilis strains. However, its application has been limited due to its relatively low transformation efficiency, a large plasmid size (13.4 kb), and limited choice of cloning sites for gene manipulations. Some of these limitations can be overcome by the newly designed and constructed plasmid pHW20a, which provides significantly higher transformation efficiency (about two orders of magnitude greater), better stability (for at least 120 generation times), and an ease of gene manipulations. The pHW20a contains three complete cis‐acting genes (repA, repB, and repC) encoding the Rep proteins for primosome formation. It has the origin of replication (oriV) to ensure replication in gram‐negative bacteria, two mob genes that enhances transformation efficiency, a screening marker (lacZα), expanded multiple cloning sites (MCS) that enables easy gene manipulation, and the tetracycline resistance gene (tcr). The utility of screening marker, lacZα with MCS, was confirmed by the blue‐white screening test. Several examples of applications of gene expression in Z. mobilis ZM4 have been demonstrated in this article by using several new pHW20a‐derived plasmids and expressing the homologous genes (gfo and ppc) and the heterologous genes (bglA, mdh, and fdh1). The results show that pHW20a is a very useful new vector for construction of new Z. mobilis recombinant strains that will enable simultaneous co‐production of biofuel and high value added products. Biotechnol. Bioeng. 2011; 108:1616–1627. © 2011 Wiley Periodicals, Inc.

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Biotechnology and Bioengineering

Tập 108 Số 7

1616-1627

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