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

Zymomonas mobilis has the potential to be an optimal chassis for the production of bulk chemicals derived from pyruvate. However, a lack of available standardized and characterized genetic tools hinders both efficient engineering of Z. mobilis and progress in basic research on this organism. In this study, a series of different shuttle vectors were constructed based on the replication mechanisms of the native Z. mobilis plasmids pZMO1, pZMOB04, pZMOB05, pZMOB06, pZMO7 and p29191_2 and on the broad host range replication origin of pBBR1. These plasmids as well as genomic integration sites were characterized for efficiency of heterologous gene expression, stability without selection and compatibility. We were able to show that a wide range of expression levels could be achieved by using different plasmid replicons. The expression levels of the constructs were consistent with the relative copy numbers, as determined by quantitative PCR. In addition, most plasmids are compatible and could be combined. To avoid plasmid loss, antibiotic selection is required for all plasmids except the pZMO7-based plasmid, which is stable also without selection pressure. Stable expression of reporter genes without the need for selection was also achieved by genomic integration. All modules were adapted to the modular cloning toolbox Zymo-Parts, allowing easy reuse and combination of elements. This work provides an overview of heterologous gene expression in Z. mobilis and adds a rich set of standardized genetic elements to an efficient cloning system, laying the foundation for future engineering and research in this area.