Cloning, Expression And Fermentation Of Bacillus Licheniformis Poly-¦Ã-glutamate Synthetase Complex Gene Regulation And Control

Poly-y-glutamic acid (y-PGA) is a kind of non-ribosomal peptide, it is made of D-and (or) L-glutamic acid units linked by amide linkages betweenα-amino and y-carboxylic acid groups with higher negative charges. It is water-soluble and biodegradable. As a friendly polymer for environment, it can be applied to various fields. In virtue of the diversity of the structure of y-PGA, the diversity of biosynthesis, the complication of metabolic regulation and the instability of y-PGA synthetase complex, up to now, agreements on its biosynthesis mechanism have not been achieved.Bacillus licheniformis WBL-3, one of poly-y-glutamic acid (y-PGA) producers, depended on the existence of glutamate in the medium. In this paper, y-PGA synthetase complex genes (pgsBCA) were cloned from B. licheniformis WBL-3 (Accession Number GQ249184). pgsBCA genes of B.licheniformis WBL-3 were highly homologous with pgsBCA genes of B. licheniformis 14580. The identity was 97%, but the identity of pgsBCA genes between B. licheniformis WBL-3 and Bacillus subtilis IFO3336 were only 74%. However, when pgsBCA genes were expressed in Escherichia coli, the E. coli clone produced y-PGA extracellularly. The yield of y-PGA was 8.624 g/L. This result infered that B. licheniformis and B. subtilis had the similar y-PGA biosynthesis mechanism, namely, glutamic acid was catalyzed by an ATP-dependent amide ligase to synthesize y-PGA.Poly-y-Glutamic Acid (y-PGA) and its derivative are a kind of new-style materials used for restoring and improving soil. They could adsorb heavy metals and radioactive nuclear materials from soil. They also could be applied to droughty soil to keep water. The aim of this paper was to research the effect of NaCl, Mn(Ⅱ), L-glutamine, and a-ketoglutarate on the production and molecular size of y-PGA. The concentration of NaCl, Mn(Ⅱ), L-glutamine, and a-ketoglutarate is 0-7%,0-400μM,0-2.0 mM and 0-20 mM respectively. No y-PGA was produced when L-glutamine and a-ketoglutarate were instead of L-glutamic acid respectively. One factor experiment indicated that the production of y-PGA was enhanced as the concentration of these four factors increased, the highest yield of y-PGA was 35.79 g/L (with 6% NaCl),24.77 g/L (with 100μM Mn(Ⅱ)),30.07 g/L (with 1.5 mM L-glutamine) and 26.09 g/L (with 10 mM a-ketoglutarate); The molecular size of y-PGA increased when the NaCl concentration increased, but decreased when the a-ketoglutarate concentration increased, the molecular size of y-PGA increased before decreasing when the Mn(Ⅱ) and L-glutamine concentration increased. The result of orthogonal experiment was in accordance with one-factor experiment. There was no interaction among these four factors. The optimal combination was 6% NaCl,10 mM a-ketoglutarate,100μM Mn(Ⅱ) and 1.5 mM L-glutamine, the highest yield was 55.62 g/L.