A 5’ fusion peptide causes heterologous over-expression: designing, cloning and testing the effect of multiple fusion peptide variants in Escherichia coli and Aliivibrio wodanis

Abstract

Since its introduction, recombinant protein production has opened an important market for industrial applications. While Escherichia coli is undoubtedly the most commonly used bacterium for protein production, its use has limits in the variety of proteins it can successfully produce. One such limitation is the production of cold-adapted proteins. Their high activity at low temperatures and potential for thermal inactivation make them interesting for industrial applications, but their low stability at the optimal growth temperatures of E. coli limits their production in this mesophilic bacterium. So, to utilise the potential of cold-adapted proteins alternative expression systems are needed. Addressing this need, the PsyXpress project was started, aiming to develop a novel expression system for cold-adapted proteins using the marine bacterium Aliivibrio wodanis. Throughout the project, expression was shown to increase when inserting the start of the AW0309160_00174 gene in front of a gene target as a 5’ fusion peptide. The mechanism behind this expression remains to be fully understood and variants designed to test the effect of fusion peptide variants on expression have previously only been tested in E. coli. This study aimed to further investigate the effect of different fusion peptide variants in A. wodanis and E. coli. With the hope of getting new insights into the mechanism that causes the increase in expression and further the development of A. wodanis as an expression host. To achieve this several fusion peptides were designed and tested in both E. coli and A. wodanis by quantifying the expression using sfGFP as a reporter protein. Based on the expression, a selection of fusion peptides was chosen to express a chitinase. The expression studies confirmed that the use of fusion peptides increased expression also in A. wodanis, however, there were observed differences in how fusion peptide variants affect expression levels in A. wodanis compared to E. coli. This indicates a difference in the translational machinery between the species. In accordance with previous studies the folding free energy of the -25 to +35 region of the promoter seemed to be important for the over-expression in E. coli. This model did not seem to be as robust for A. wodanis. There was also observed an indication that the AT content of the fusion peptide has a higher impact on the expression in A. wodanis, however, due to variation between parallels and few replicates, further investigation is needed to draw conclusions. Overall, the study gave new insight into A. wodanis as an expression system and the considerations that should be taken when using it to express cold-adapted proteins.