Engineering of Aminotransferase for Green Biosynthesis of Glucosidase Inhibitor Valienamine in <i>Streptomyces hygroscopicus</i> 5008

Valienamine is a d-glucose analogue with potent inhibitory activity toward glucosidase that is used as a precursor in the development of antidiabetic drugs. An artificial transamination reaction using WecE on valienone could address the challenge of creating multiple chiral centers in valienamine. However, the low catalytic activity of WecE hindered valienamine production. Here, we present a rational steric hindrance-leveraging-based interaction modification strategy that included structural and interaction analysis as well as tryptophan steric hindrance scanning mutagenesis to enhance the catalytic activity of the previously obtained mutant of WecE (M0). We identified and iterated three new functional residues, resulting in a triple mutant of M0, K209W/K25N/I322D, named M3. M3 exhibited a nearly 6-fold improvement in activity compared with M0. By applying metabolic engineering strategies to integrate and overexpress M3, a valienamine synthesis pathway was reconstructed in Streptomyces hygroscopicus strain 5008. The resulting strain yielded 2.6 mg/L valienamine after 96 h of culture, nearly 5 times that from the strain engineered using M0. The approach demonstrated an efficient strategy here that is useful for more economic production of the valuable compound valienamine and is generalizable to improve enzymatic reactions of non-natural small substrates in an oversized binding pocket.