Aliivibrio wodanis as a production host: development of genetic tools for expression of cold-active enzymes
Results: We tested 12 bacterial strains, as well as available vectors, promoters and reporter systems. We used RNAsequencing to determine the most highly expressed genes and their intrinsic promoters in A. wodanis. In addition we examined a novel 5′-fusion to stimulate protein production and solubility. Finally we tested production of a set of “difcult-to-produce” enzymes originating from various bacteria and one Archaea. Our results show that coldadapted enzymes can be produced in soluble and active form, even in cases when protein production failed in E. coli due to the formation of inclusion bodies. Moreover, we identifed a 60-bp/20-aa fragment from the 5′-end of the AW0309160_00174 gene that stimulates expression of Green Fluorescent Protein and improves production of coldactive enzymes when used as a 5′-fusion. A 25-aa peptide from the same protein enhanced secretion of a 25-aa-sfGFP fusion.
Conclusions: Our results indicate the use of A. wodanis and associated genetic tools for low-temperature protein production and indicate that A. wodanis represents an interesting platform for further development of a protein production system that can promote further cold-enzyme discoveries.