Aims Poly-gamma-glutamic acid (gamma-PGA) is an excellent water-soluble biosynthesis material. To confirm the rate-limiting steps of gamma-PGA biosynthesis pathway, we introduced a heterologous Bacillus strain pathway and employed an enzyme-modulated dismemberment strategy in Escherichia coli. Methods and Results In this study, we heterologously introduced the gamma-PGA biosynthesis pathway of two laboratory-preserved strains-Bacillus amyloliquefaciens FZB42 and Bacillus subtilis 168 into E. coli, and compared their gamma-PGA production levels. Next, by changing the plasmid copy numbers and supplying sodium glutamate, we explored the effects of gene expression levels and concentrations of the substrate l-glutamic acid on gamma-PGA production. We finally employed a two-plasmid induction system using an enzyme-modulated dismemberment of pgsBCAE operon to confirm the rate-limiting genes of the gamma-PGA biosynthesis pathway. Conclusion Through heterologously over-expressing the genes of the gamma-PGA biosynthesis pathway and exploring gene expression levels, we produced 0 center dot 77 g l(-1) gamma-PGA in strain RSF-EBCAE(BS). We also confirmed that the rate-limiting genes of the gamma-PGA biosynthesis pathway were pgsB and pgsC. Significance and Impact of the Study This work is beneficial to increase gamma-PGA production and study the mechanism of gamma-PGA biosynthesis enzymes.