%0 Journal Article
%T Engineering Glucosamine-6-Phosphate Synthase to Achieve Efficient One-Step Biosynthesis of Glucosamine.
%A Guan ZB
%A Deng XT
%A Zhang ZH
%A Xu GC
%A Cheng WL
%A Liao XR
%A Cai YJ
%J ACS Chem Biol
%V 19
%N 6
%D 2024 Jun 21
%M 38723147
%F 4.634
%R 10.1021/acschembio.4c00144
%X As an important functional monosaccharide, glucosamine (GlcN) is widely used in fields such as medicine, food nutrition, and health care. Here, we report a distinct GlcN biosynthesis method that utilizes engineered Bacillus subtilis glucosamine-6-phosphate synthase (BsGlmS) to convert D-fructose to directly generate GlcN. The best variant obtained by using a combinatorial active-site saturation test/iterative saturation mutagenesis (CAST/ISM) strategy was a quadruple mutant S596D/V597G/S347H/G299Q (BsGlmS-BK19), which has a catalytic activity 1736-fold that of the wild type toward D-fructose. Upon using mutant BK19 as a whole-cell catalyst, D-fructose was converted into GlcN with 65.32% conversion in 6 h, whereas the wild type only attained a conversion rate of 0.31% under the same conditions. Molecular docking and molecular dynamics simulations were implemented to provide insights into the mechanism underlying the enhanced activity of BK19. Importantly, the BsGlmS-BK19 variant specifically catalyzes D-fructose without the need for phosphorylated substrates, representing a significant advancement in GlcN biosynthesis.