Abstract

Background: The ribosome binding site (RBS) containing Shine-Dalgarno (SD) sequence in prokaryotes plays an important role in identification of the translation initiation site within mRNA by ribosome.

Objective: Our study aimed to improve the target protein production under translational level in prokaryotes by engineering of RBSs.

Method: Two RBSs were designed in Streptomyces coelicolor M145, Sco-RBS* with an SD sequence which is completely complementary to 3'-end of 16S RNA and Sco-RBS0 with an SD sequence which is completely non-complementary to 3'-end of 16S RNA. The enhanced green fluorescent protein (EGFP) was used as a reporter, whose gene was transcribed and translated under the control of Sco- RBS*, Sco-RBS0, and a native RBS Sco-RBSactI in recombinant S. coelicolor strains.

Results: Replacement of Sco-RBSactI with Sco-RBS* resulted in increase of both EGFP production and the ratio of EGFP to EGFP mRNA by 2.67-fold and 6.07-fold, respectively. Replacement of Sco- RBSactI with Sco-RBS0 resulted in decrease of both EGFP and the ratio of EGFP to EGFP mRNA by 4.35-fold and 2.18-fold, respectively.

Conclusion: We provided a method to increase protein production at the translational level in Streptomyces.

Keywords: Binding site, EGFP production, enhanced green fluorescent protein, recombinant strains, ribosome, Streptomyces coelicolor.

Graphical Abstract