Synthesis of poly (γ-glutamic acid) and heterologous expression of pgsBCA genes

The genes required for synthesis of poly (γ-glutamic acid) (γ-PGA) were cloned from Bacillus licheniformis NK-03, a strain isolated from fermented food, natto. There were three open reading frames pgsB, pgsC, pgsA in the cloned fragment, all of which were greatly similar with those from typical Bacillus subtilis strains. The alignment of deduced amino acid sequences showed that PgsC was the most conservative part in PgsBCA. Recombinant plasmid pXMJ19-PGS was constructed by a shuttle vector pXMJ19, and it was successfully transformed and expressed in the recombinant strains of Escherichia coli JM109 and Corynebacterium glutamicum ATCC13032, respectively. Expression of pgsBCA in C. glutamicum indicated that it could synthesize γ-PGA with a yield of 0.69 g/L and 97% proportion of l-glutamate monomer in the absence of glutamic acid. The results suggest that γ-PGA biosynthesis directly from glucose by genetic engineering is feasible and significant.

Graphical abstractFigure optionsDownload full-size imageDownload as PowerPoint slideResearch highlightsγ-PGA is an unusual macromolecular anionic polypeptide, which can be widely used in various potential fields by the characteristic of water soluble, biodegradable, nontoxic, edible, etc. At present, microbial γ-PGA was mostly produced from glutamic acid dependent strains, and consist of more d-glutamate monomer. Although the γ-PGA synthetase genes (pgsBCA) had already studied for about twenty years, they were cloned confined in Bacillus subtilis and expressed in Escherichia coli. In this research, we cloned pgsBCA from a newly glutamic acid dependent γ-PGA producing strain, Bacillus licheniformis NK-03, and tried to express the genes in E. coli and a glutamic acid producer Coryneform bacteria by a shuttle vector pXMJ19. Expression of pgsBCA in C. glutamicum ATCC13032 indicated that it could synthesize γ-PGA with a yield of 0.69 g/L and 97% proportion of l-glutamate monomer in the absence of glutamic acid. The results suggest that it is promising for the synthesis of l-isomer-rich PGA directly from glucose by genetic engineering.