Abstract: This study is aimed to establish an effective tool to control gene expression in Gluconobacter oxydans. Cis- or trans-acting non-coding small RNAs，complementary to ribosomal binding site（RBS），were designed and constructed to modulate gene expression in G. oxydans at translational level. Using green fluorescent protein as reporter gene，in a cis-acting configuration，a cis repressor sequence inserted into the RBS upstream bound with RBS to form a stem-loop structure in mRNA，which repressed the reporter gene expression. Such a stem-loop was disrupted by an anti-repressor sequence that had higher affinity than a cis repressor sequence，thus this allowed the reporter gene to re-initiate translation. In a trans-acting configuration，the complementary double strands of small non-coding RNA transcribed from a separate promoter and a RBS were able to specifically repress the reporter gene expression by blocking the binding of ribosome and mRNA. Based on the above results，a series of trans-acting small RNAs were designed and imported，by which the endogenous gene pstI expression inhibition in G. oxydans was realized. In summary，this study offers an alternative approach to study gene functions and regulate gene expression in G. oxydans other than gene knockout.

Key words: Gluconobacter oxydans, small non-coding RNA, translational regulation, ribosome binding site, gene knockout