Engineering\nof Aminotransferase for Green Biosynthesis\nof Glucosidase Inhibitor Valienamine in Streptomyces\nhygroscopicus 5008

Valienamine is a d-glucose analogue with potent\ninhibitory\nactivity toward glucosidase that is used as a precursor in the development\nof antidiabetic drugs. An artificial transamination reaction using\nWecE on valienone could address the challenge of creating multiple\nchiral centers in valienamine. However, the low catalytic activity\nof WecE hindered valienamine production. Here, we present a rational\nsteric hindrance-leveraging-based interaction modification strategy\nthat included structural and interaction analysis as well as tryptophan\nsteric hindrance scanning mutagenesis to enhance the catalytic activity\nof the previously obtained mutant of WecE (M0). We identified and\niterated three new functional residues, resulting in a triple mutant\nof M0, K209W/K25N/I322D, named M3. M3 exhibited a nearly 6-fold improvement\nin activity compared with M0. By applying metabolic engineering strategies\nto integrate and overexpress M3, a valienamine synthesis pathway was\nreconstructed in Streptomyces hygroscopicus strain 5008. The resulting strain yielded 2.6 mg/L valienamine after\n96 h of culture, nearly 5 times that from the strain engineered using\nM0. The approach demonstrated an efficient strategy here that is useful\nfor more economic production of the valuable compound valienamine\nand is generalizable to improve enzymatic reactions of non-natural\nsmall substrates in an oversized binding pocket.