Improving the Synthesis Efficiency of Amino Acids Such as L-Lysine by Assembling Artificial Cellulosome Elements Dockerin Protein In Vivo

Cellulosome is a highly efficient multi-enzyme self-assembly system and is found on the extracellular surface or in the free environment of microorganisms. However, with a lack of Ca2+ in vivo, cellulosome assembly is challenging. In this study, a novel design method was used to directionally modify the Ca2+-binding site, and four double-site dockerin A (DocA) mutants were obtained. At a Ca2+ concentration between 1.00 × 10−7 and 1.00 × 10−4 M, the mutant DocA-D3 had the strongest binding capacity to cohesion (Coh), which was 8.01 times that of DocA. The fluorescence signal intensity of the fusion proteins assembled using mutants was up to 1.26 × 107 in Escherichia coli, which indicated that these mutants could interact with Coh in vivo. The molecular dynamics simulation results showed that DocA-D3 could maintain a stable angle structure without Ca2+, and when applied to L-lysine fermentation, the yield was increased by 24.1%; when applied to β-alanine fermentation, the product accumulation was increased by 2.13–2.63 times. These findings lay the foundation for assembly design in cells.