Codon Optimization is Required to Express Fluorogenic Reporter Proteins in Lactococcus lactis

Lactococcus lactis is a Gram-positive bacterium used to produce fermented foods and heterologous proteins. Its Nisin-controlled gene expression system stands out for its versatility and safety. However, the lower GC content in its genome may lead to some limitations in protein production. In this study, we explored the importance and effect of codon optimization on fluorescent reporter protein expression in L. lactis. Three non-optimized fluorescent reporter genes (gfp, rfp, and mcherry) were compared to the codon-optimized variant (mcherry-O). Parameters such as Codon Adaptation Index (CAI), Effective Number of Codons (Enc) and Guanine-Cytosine percentage (% GC) were determined to assess their influence on gene expression and protein synthesis. The production of non-optimized fluorescent proteins does not correlate with their gene expression levels, except for the codon-optimized mCherry-O protein, which was detected in the SDS-PAGE gel and the extracted lysate (visually detected). Expression of the mcherry gene was similar to the mcherry-O gene, but protein was only detected with the optimized gene. The gfp gene showed the highest expression levels, but the quantity of protein was undetectable by SDS-PAGE. The rfp gene was revealed to be an optimized gene but not tailored for L. lactis. These findings underscore the necessity of comprehensive codon optimization for foreign genes in L. lactis and reveal intriguing complexities between expression levels, RNA stability and protein synthesis.