Elucidating the regulatory role of the Shine-Dalgarno sequence in structural gene expression of the malolactic enzyme...

Malolactic fermentation (MLF) in wine relies on Oenococcus oeni, in which the mleA and mleP genes are translationally coupled. To explore the post-transcriptional regulation involved, we constructed parallel GFP/mCherry reporter systems that mimic both the native overlapping (translationally coupled) structure and an engineered non-overlapping (non-coupled) structure. Through site-directed SD2 mutagenesis coupled with computational prediction of 16 S rRNA hybridization energy (ΔG) and minimum free energy (MFE) of mRNA secondary structure, we quantified the effect of intergenic sequence features on translation efficiency. In the translational coupling system of Escherichia coli, expression ratios (GFP/MCH) varied from 3.58 to 8.58 across variants, while in the non-coupled system, they ranged from 1.54 to 3.09. In non-coupled constructs, downstream gene expression increased under independent SD2 control, consistent with its conserved function. However, in the coupling system, the strongest SD2 (GGAGG) markedly suppressed it, revealing a nonlinear, architecture-specific regulatory logic. The different variants were also expressed in the translational coupling system of Lactococcus lactis, and the expression ratio patterns of the upstream and downstream genes across the variants were consistent with those observed in E. coli. This indicated that SD2 had the same regulatory effect on coupling gene expression ratios in both L. lactis and E. coli. Our findings demonstrate that translational coupling establishes precise protein expression regulation through the integrated effects of upstream translation and local mRNA structure. This work elucidates a key post-transcriptional tuning layer in prokaryotic operons, providing a predictive framework for both the rational engineering of O. oeni to optimize MLF and the design of proportionally controlled multi-gene systems in synthetic biology.