Precision Quantificationand Rational Regulation ofProtein Expression with Bicistronic Cassette for Efficient BiotinProduction

Identifying optimal enzyme expression levels is critical for microbial cell factories, as metabolic imbalances can impede the synthesis of target products. However, current screening strategies often rely on trial-and-error approaches, which are labor-intensive and have limited applicability. Here we developed a quantitative strategy utilizing a bicistronic design (BCD) library for enzyme expression screening, requiring no more than 17 tests in two steps: expression profiling and focused selection. The BCD library encoded a 992-fold expression range, and protein abundances were quantified based on fluorescence intensities due to a strong correlation (<i>r</i> = 0.96). This strategy was employed to fine-tune the expression of the rate-limiting enzyme BioB in biotin synthesis, whose overexpression inhibits cell growth and biotin production. Consequently, BCD6 was identified the optimal expression strength for the overexpressed <i>bio</i> operon, while BCD7 was optimal for the overexpressed <i>bio</i> + <i>isc</i> operons, resulting in 1.47-fold and 3.03-fold increases in biotin titer compared to original strain. Western Blot analysis confirmed a 2.38-fold and 2.71-fold increase in BioB abundance, respectively. The pioneering application of BCD establishes it as a versatile tool for the rational tuning of enzyme expression in the construction of any microbial cell factory.