Background: Pullulanase production in both wild-type strains and recombinantly engineered strains remains low. The Shine-Dalgarno (SD) sequence and stem-loop structure in the 5′ or 3′ untranslated region (UTR) are well-known determinants of mRNA stability. This study investigated the effect of mRNA stability on pullulanase heterologous expression.

Results: We constructed four DNA fragments, pulA, SD-pulA, pulA-3t, and SD-pulA-3t, which were cloned into the expression vector pHT43 to generate four pullulanase expression plasmids. The DNA fragment pulA was the coding sequence (CDS) of pulA in Klebsiella variicola Z-13. SD-pulA was constructed by the addition of the 5′ SD sequence at the 5′ UTR of pulA. pulA-3t was constructed by the addition of a 3′ stem-loop structure at the 3′ UTR of pulA. SD-pulA-3t was constructed by the addition of the 5′ SD sequence at the 5′ UTR and a 3′ stem-loop structure at the 3′ UTR of pulA. The four vectors were transformed into Escherichia coli BL21(DE3). The pulA mRNA transcription of the transformant harboring pHT43-SD-pulA-3t was 338.6%, 34.9%, and 79.9% higher than that of the other three transformants, whereas the fermentation enzyme activities in culture broth and intracellularly were 107.0 and 584.1 times, 1.2 and 2.0 times, and 62.0 and 531.5 times the amount of the other three transformants (pulA, SD-pulA, and pulA-3 t), respectively.

Conclusion: The addition of the 5′ SD sequence at the 5′ UTR and a 3′ stem-loop structure at the 3′ UTR of the pulA gene is an effective approach to increase pulA gene expression and fermentation enzyme activity.