Rational Modification of Polyphosphate-Dependent Mannose Kinase for Efficient Synthesis of Mannose-6-Phosphate

Abstract

Microbial heterologous biosynthesis of natural products often faces challenges due to the inhibitory effects of enzyme products. This study addresses the feedback inhibition of mannose-6-phosphate (M6P) on polyphosphate-dependent mannose kinase by employing a combinatorial strategy. Directed evolution of AsPPGMK enhances its activity, coupled with the optimization of whole-cell reaction conditions to increase M6P production. A new high-throughput screening method was used for the directed evolution of AsPPGMK, and a dual mutant L169I/I174L (Mu5) was obtained with a conversion of 93.5%. Computational analysis indicated an enhancement in the hydrogen bond network of the Mu5 active center. Optimization of biotransformation reaction conditions resulted in a remarkable 96% conversion, reaching a record-high production of 499.7 g L-1 of M6P. This achievement represents the highest reported yield after relieving substrate product inhibition by new mutant enzyme. The study introduces a new mutant enzyme and an efficient synthesis technology, significantly reducing preparation costs. This work establishes a robust foundation for industrial-scale M6P production using protein engineering and gene engineering.

Keywords: Mannose-6-phosphate, Polyphosphate dependent kinase, Directed evolution, High throughput screening, Computational analysis