Overcoming hydrophobicity-driven aggregation: An integrated expression strategy enables high-yield soluble production...

Diverse enzyme resources are essential for advancing cell-free chemoenzymatic starch synthesis from CO₂. However, heterologous expression of such enzymes in Escherichia coli often leads to protein misfolding and inclusion body formation, resulting in low yields of soluble, active proteins and limiting large-scale applications. Here, starch synthase from Chloracidobacterium thermophilum (CtSS) was selected as a model enzyme because it was predicted to be thermostable yet aggregation-prone. Structural analysis revealed a highly hydrophobic surface and multiple flexible loop regions that likely contribute to aggregation. Based on these insights, we developed an integrated optimization strategy. MBP fusion combined with optimized induction conditions was the most effective approach for improving CtSS solubility, resulting in an 8.1-fold increase in soluble expression. In addition, removal of the MBP tag from CtSS increased catalytic activity by 4.5-fold. To assess the broader applicability of this strategy, we applied codon optimization for E. coli, MBP fusion, and optimized induction conditions to four phylogenetically distinct starch biosynthetic enzymes: phosphoglucomutase from Glycine max (GmPGM), ADP-glucose pyrophosphorylase from Zea mays (ZmAGP), and starch synthases from Oryza sativa (OsSS) and Manihot esculenta (MeSS). All four enzymes showed improved soluble expression, with increases of 3.0-, 3.1-, 3.7-, and 9.5-fold, respectively. This study establishes an integrated strategy for improving the soluble expression of starch biosynthetic enzymes in prokaryotic hosts, providing methodological support for the optimization of artificial starch anabolic pathways and a useful framework for the heterologous expression of structurally complex proteins in synthetic biology and industrial applications.