Whole genome sequencing of bacillus subtilis Natto3 and cloning and expression of ywtA, a pivotal gene of poly-γ-glutamic acid biosynthesis operon

Poly-γ-glutamic acid (PGA) is produced via PGA synthetase, a membrane-associated enzyme complex encoded by pgs operon consisting of ywsC and ywtABC genes. In our earlier report, we had studied the unique expression pattern of pgs operon in Bacillus subtilis (B. subtilis) Natto3 strain. This motivated us to perform whole genome sequencing of this strain and analyse the sequence of the genes involved in PGA biosynthesis. B. subtilis Natto3 genome has 3,637,088 bp with 44.08% G-C content and no plasmid. It contains 3913 coding sequences (CDSs) with average gene length of 843.33 bp with 57 CDSs lacking matches. A recent study emphasised the conserved nature of ywtA gene in pgs operon. This gene may provide further insights into the role of individual genes in PGA biosynthesis. In this study, we have successfully cloned the ywtA gene in E. coli DH5α and expressed in BL21 (DE3) strain. The purified protein was ~16 kDa which was in congruence with the existing studies. Protein identification using Nano LC-MS/MS showed homology to CapC protein of B. anthracis with a match of 15%.