Relieving Allosteric Inhibition by Designing Active Inclusion Bodies and Coating of the Inclusion Bodies with Fe3O4 Nanomaterials for Sustainable 2-Oxobutyric Acid Production

Allosteric inhibition of key enzymes by end-product has been widely studied in control of valuable compound biosynthesis, and site-directed mutagenesis is often applied for relieving allosteric inhibition. Here we rationally designed a different approach to relieve allosteric inhibition by Ile in threonine deaminase (TD) pathway and efficiently produced 2-oxobutyric acid in a save-energy way. We truncated the different peptide length at C-terminal regulatory domain (from residue 11 to 193) Escherichia coli TD and obtained the active inclusion bodies. The truncated residues Glu480-Gly514 in C-terminal regulatory domain were confirmed to be an unstable structure by MD simulations. The truncation variants Δ11, Δ25 and Δ35 weren’t allosterically regulated by Ile at all by kinetics analysis. They showed decreased activity but better thermostability than wild-type enzyme. Among the three variants, Δ25 showed 3.3-fold higher 2-oxobutyric acid production in the presence of 2 mM Ile and about 9-fold higher product...