Introducing glutamic acid residues to acyl-ACP reductase to enhance alka(e)ne production in Escherichia coli: Computer-aided design and subsequent experimental validation

Acyl-acyl carrier protein (acyl-ACP) reductase (AAR) is a crucial enzyme in alka(e)ne production by recombinant Escherichia coli (E. coli). Engineered AAR expressed in E. coli holds great promise for the production of alka(e)nes, which are a valuable bio-based alternative to fossil fuels. However, its effectiveness is significantly limited by its low solubility and stability. The aim of this study is to enhance the solubility and stability of AAR to improve the production of alka(e)nes in E. coli. In this study, an integrated computational approach was employed for combining solubility prediction, aggregation propensity prediction, structural modeling, and molecular dynamics (MD) simulations. This multi-faceted approach provides new insights and tools for enzyme engineering. Through this approach, the C-terminus of AAR was identified as the sole significant hydrophobic patch and aggregation-prone regions (APR). Three strategies were evaluated experimentally: direct deletion of these hydrophobic residues; substitution of these residues with negatively charged amino acids, such as glutamic acid (Glu) or aspartic acid (Asp); and the introduction of additional negatively charged amino acids at the C-terminus to shield the hydrophobic patches. The results showed that AAR mutants with additional Glu residues at the C-terminus exhibited improved performance. Specifically, the AAR-E₃ mutant, containing three consecutive Glu residues, demonstrated significantly enhanced solubility and stability, with alka(e)ne production (159.25 mg/L) being 6.3 times higher than that of the wild-type AAR (25.37 mg/L). Subsequent computational modeling and molecular dynamics simulations further validated the experimental findings. This study highlights the potential of enzyme engineering to significantly enhance biofuel production efficiency.