ena-STUDY-University of Tromso-10-01-2019-08:14:00:971-731
In this work we explored the use of the sub-Arctic bacterium Aliivibrio wodanis as a potential host for heterologous expression of cold-active enzymes.
Background: Heterologous expression of cold-adapted proteins represent today one of the biggest bottlenecks in the ongoing bioprospecting efforts to find new enzymes from cold environment, e.g., the cold oceans that represents largely untapped resources in this respect. In mesophilic expression hosts such as Escherichia coli, cold-adapted enzymes typically form inactive aggregates. It is therefore a need to develop new cold-temperature expression systems, including identifying new host organisms and genetic tools. Psychrophilic bacteria, including Pseudoalteromonas haloplanctis, Schewanella and Rhodococcus erythropolis have all been explored for such use, but none of them are commercially available, or are available for common use as efficient expression systems. In this work we explored the use of the sub-Arctic bacterium Aliivibrio wodanis as a potential host for heterologous expression of cold-active enzymes.Results: We have tested twelve bacterial strains, available vectors and promoters, reporter systems, used RNA-sequencing to identify the most highly expressed genes in A. wodanis (and thus their intrinsic promoters), explored a novel 5'-fusion to stimulate protein expression and solubility, and finally tested expression of a set of “difficult-to-express” enzymes originating from various bacteria and one Archaea. Our results show that cold-adapted enzymes can be expressed in soluble and active form, also when expression fails in E. coli due to formation of inclusion bodies. Moreover, we identified a 60-bp/20-aa fragment from the 5'-end of the Awod_I1781 gene that stimulates expression of the Green Fluorescent Protein and improves expression of cold-active enzymes when used as a 5'-fusion. Conclusions: Our results support that A. wodanis and associated genetic tools are well suited for low-temperature expression, and that A. wodanis represent an interesting platform for further developments of an expression systems that can promote further cold-enzyme discoveries.
Background: Heterologous expression of cold-adapted proteins represent today one of the biggest bottlenecks in the ongoing bioprospecting efforts to find new enzymes from cold environment, e.g., the cold oceans that represents largely untapped resources in this respect. In mesophilic expression hosts such as Escherichia coli, cold-adapted enzymes typically form inactive aggregates. It is therefore a need to develop new cold-temperature expression systems, including identifying new host organisms and genetic tools. Psychrophilic bacteria, including Pseudoalteromonas haloplanctis, Schewanella and Rhodococcus erythropolis have all been explored for such use, but none of them are commercially available, or are available for common use as efficient expression systems. In this work we explored the use of the sub-Arctic bacterium Aliivibrio wodanis as a potential host for heterologous expression of cold-active enzymes.Results: We have tested twelve bacterial strains, available vectors and promoters, reporter systems, used RNA-sequencing to identify the most highly expressed genes in A. wodanis (and thus their intrinsic promoters), explored a novel 5'-fusion to stimulate protein expression and solubility, and finally tested expression of a set of “difficult-to-express” enzymes originating from various bacteria and one Archaea. Our results show that cold-adapted enzymes can be expressed in soluble and active form, also when expression fails in E. coli due to formation of inclusion bodies. Moreover, we identified a 60-bp/20-aa fragment from the 5'-end of the Awod_I1781 gene that stimulates expression of the Green Fluorescent Protein and improves expression of cold-active enzymes when used as a 5'-fusion. Conclusions: Our results support that A. wodanis and associated genetic tools are well suited for low-temperature expression, and that A. wodanis represent an interesting platform for further developments of an expression systems that can promote further cold-enzyme discoveries.