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Poly-gamma-glutamic acid (PGA) is a promising bio-based polymer that shares many functions with poly (acrylic acid) and its derivatives. Thus, technologies for efficient production and molecular size control of PGA are required to expand the application of this useful biopolymer. In  Bacillus  strains, PGA is synthesized by the PgsBCA protein complex, which is encoded by the  pgsBCA  gene operon, otherwise is known as  ywsC  and  ywtAB  operons and/or  capBCA  operon. Hence, we investigated responsible components of the PgsBCA complex in  B. subtilis  for over-production of PGA. In particular, we constructed genomic  pgsBCA  gene-deletion mutants of  B .  subtilis . And also, we assembled high copy-number plasmids harboring σA-dependent promoter, leading to high-level expression of all combinations of  pgsBCA ,  pgsBC ,  pgsBA ,  pgsCA ,  pgsB ,  pgsC , and/or  pgsA  genes. Subsequently, PGA production of the transformed  B .  subtilis  mutant was determined in batch fermentation using medium supplemented with  l -glutamate. PGA production by the transformants introduced with  pgsBC  genes (lacking the genomic  pgsBCA  genes) was 26.0 ± 3.0 g L −1 , and the enantiomeric ratio of  d - and  l -glutamic acid ( d / l -ratio) in the produced PGA was 5/95. In contrast,  d / l -ratio of produced PGA by the transformants introduced with  pgsBCA  genes (control strains) was 75/25. In conclusion,  B .  subtilis  without  pgsA  gene could over-produce PGA with an  l -rich enantiomeric ratio.

Poly-l-gamma-glutamic acid production by recombinant Bacillus subtilis without pgsA gene