pZMO7-Derived shuttle vectors for heterologous protein expression and proteomic applications in the ethanol-producing bacterium Zymomonas mobilis

BMC Microbiology

Rory M Watt³  Myriam Seemann¹  Donnabella C Lacap-Bugler⁴  Wen-yang Chen³  Lok Yan So² 

[1]Université de Strasbourg, CNRS UMR 7177, Institut Le Bel, 4 rue Blaise Pascal, CS 90032, Strasbourg 67081Cedex, France

[2]Present Address: Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong

[3]Oral Biosciences Faculty of Dentistry, The University of Hong Kong, 34 Hospital Road, Sai Ying Pun, Hong Kong

[4]Oral Diagnosis and Polyclinics, Faculty of Dentistry, The University of Hong Kong, Prince Philip Dental Hospital, 34 Hospital Road, Sai Ying Pun, Hong Kong

关键词: Microbial biotechnology;    Affinity purification;    Proteomics;    Quantitative PCR;    Replication;    Shuttle vector;    Plasmid;    Zymomonas mobilis;   

Others  :  1141648
DOI  :  10.1186/1471-2180-14-68

 received in 2013-05-06, accepted in 2014-03-06,  发布年份 2014

【 摘 要 】

Background

The ethanol-producing bacterium Zymomonas mobilis has attracted considerable scientific and commercial interest due to its exceptional physiological properties. Shuttle vectors derived from native plasmids have previously been successfully used for heterologous gene expression in this bacterium for a variety of purposes, most notably for metabolic engineering applications.

Results

A quantitative PCR (qPCR) approach was used to determine the copy numbers of two endogenous double stranded DNA plasmids: pZMO1A (1,647 bp) and pZMO7 (pZA1003; 4,551 bp) within the NCIMB 11163 strain of Z. mobilis. Data indicated pZMO1A and pZMO7 were present at ca. 3-5 and ca. 1-2 copies per cell, respectively. A ca. 1,900 bp fragment from plasmid pZMO7 was used to construct two Escherichia coli – Z. mobilis shuttle vectors (pZ7C and pZ7-184). The intracellular stabilities and copy numbers of pZ7C and pZ7-184 were characterized within the NCIMB 11163, ATCC 29191 and (ATCC 10988-derived) CU1 Rif2 strains of Z. mobilis. Both shuttle vectors could be stably maintained within the ATCC 29191 strain (ca. 20-40 copies per cell), and the CU1 Rif2 strain (ca. 2-3 copies per cell), for more than 50 generations in the absence of an antibiotic selectable marker. A selectable marker was required for shuttle vector maintenance in the parental NCIMB 11163 strain; most probably due to competition for replication with the endogenous pZMO7 plasmid molecules. N-terminal glutathione S-transferase (GST)-fusions of four endogenous proteins, namely the acyl-carrier protein (AcpP); 2-dehydro-3-deoxyphosphooctonate aldolase (KdsA); DNA polymerase III chi subunit (HolC); and the RNA chaperone protein Hfq; were successfully expressed from pZ7C-derived shuttle vectors, and their protein-protein binding interactions were analyzed in Z. mobilis ATCC 29191. Using this approach, proteins that co-purified with AcpP and KdsA were identified.

Conclusions

We show that a shuttle vector-based protein affinity ‘pull-down’ approach can be used to probe protein interaction networks in Z. mobilis cells. Our results demonstrate that protein expression plasmids derived from pZMO7 have significant potential for use in future biological or biotechnological applications within Z. mobilis.

【 授权许可】

   
2014 So et al.; licensee BioMed Central Ltd.

【 图 表 】

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