Multiple Regulatory Mechanisms Synergistically Control the Soluble Expression of <i>Cs</i>CE for Enhanced Enzymatic Productivity of Lactulose in <i>E. coli</i>

The limited expression of cellobiose 2-epimerase poses a significant constraint on the industrial enzymatic production of lactulose. Extensive modifications to the expression cassette offer a means to enhance the yield of recombinant proteins. In this study, an integrated strategy, combining individual and collaborative approaches, is proposed to fine-tune each stage of the <i>Cs</i>CE overexpression program. This strategy involves the multidimensional integration of standardized genetic elements at various levels, including transcription, translation, folding, and three-dimensional structure. The volumetric activity of the final recombinant strain was markedly increased by 12-fold compared to the wild-type strain, reaching 2260.62 U/L. The protein expression in the newly developed high-yield recombinant strain exhibited a significant enhancement, with a higher proportion of soluble protein compared to that of inclusion bodies. Our findings offer insights into the multifaceted synergistic regulation of protein expression processes, holding promising implications for the production of heterologous recombinant proteins.