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

Abstract

BackgroundThe 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.ResultsA 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.ConclusionsWe 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.

Construction and comparison of different vehicles for heterologous gene expression in Zymomonas mobilis

“…For pZP605 even after 50 generations without antibiotic selection, almost all cells in the population expressed mCherry. This confirms the observation by So et al (2014) who reported that their pZMO7-based shuttle vector was stable for at least 50 generations. pZP605, unlike the derivative pZ7C (So et al, 2014), does not carry any of the genes of pZMO7 other than the putative replication-associated gene.…”

Using the CRISPR/Cas9 system to eliminate native plasmids of Zymomonas mobilis ZM4

“…The cell pellets were treated at 95°C for 10 min followed by immediate freezing at −20°C and used as the templates for quantitative (real time) PCR analyses. 23,24) DNA concentrations were determined using a Nanodrop 2000 spectrophotometer (Thermo Scientific, DE, USA). qPCR was performed using an iQ5 Real-time PCR System (Bio-Rad Laboratories, Inc., Hercules, CA, USA) with reaction mixtures (total volume 20 μL) containing 10 μL 2 × TaKaRa SYBR Green Real-Time PCR Master Mix (Takara, Dalian, China), 1.5 μL forward and reverse primers (10 μM stock), and 1 μL of sample.…”

Zymomonas mobilis as an emerging biotechnological chassis for the production of industrially relevant compounds

“…E. coli-Z. mobilis shuttle vectors, which contain replicons from E. coli and from Z. mobilis native plasmids, are currently the most popular plasmid-based expression systems (So et al 2014) (Fig. 3A).…”