[3S04a] 11. The Society for Biotechnology - Part 2

2:50 PM - 3:10 PM JST(5:50 AM - 6:10 AM UTC)

[3SBT-10] Engineering polymer stereochemistry to enable high-molecular-weight poly-γ-glutamate production in Bacillus subtilis

○Shu Ishikawa¹, Onuma Chumsakul¹ (1. Kobe University (Japan))

Keywords:

Poly-γ-glutamate,Bacillus subtilis,Polymer stereochemistry,Epimerase,Biopolymer production

Poly-γ-glutamate (PGA) is a biodegradable microbial biopolymer whose physicochemical and material properties strongly depend on its D/L stereochemistry. In Bacillus species, PGA is typically produced as DL-PGA, whereas stereoregular L-PGA exhibits distinct functional properties and enhanced stability, making it attractive for advanced bio-based materials. In this study, we investigated the molecular basis of stereochemical control in PGA biosynthesis in Bacillus subtilis, focusing on the PGA synthase subunit PgsA. Through analyses of spontaneous mutants, systematic truncation variants, and targeted mutagenesis, we demonstrate that PgsA performs two separable functions in vivo: the N-terminal cytosolic and transmembrane region is indispensable for productive PGA synthesis, whereas the extracytoplasmic C-terminal domain functions as an epimerase that introduces D-glutamate residues into the polymer. Disabling this epimerase activity through specific amino-acid substitutions converts DL-PGA synthesis to predominantly L-PGA while maintaining polymer production. Because endogenous hydrolases preferentially cleave D-containing linkages, this stereochemical switch reduces post-synthetic degradation, resulting in substantially higher apparent molecular weight polymers. These findings establish stereochemical control of PGA biosynthesis as a practical engineering strategy for producing high-molecular-weight, hydrolysis-resistant L-PGA in a standard Bacillus chassis.