Effects On Producing Poly-γ-glutamate By Enhancing Expression Of YwtD Gene In Bacillus Licheniformis

Poly-y-glutamic acid (y-PGA) is one of the most promising biodegradable polymer that has potential applications in a wide range of food, cosmetics, medicinal industries and agriculture. With the production of y-PGA, y-PGA fermentation broths exhibit high viscosity and non-Newtonian rheology, which limits y-PGA fermentation and downstream purification. In addition, since different molecular weight of y-PGA is requested for different purposes, the control of it is important for its application.γ-PGA depolymerase serves as a potent tool for such purposes. The reports of y-PGA depolymerase are major in enzymatic properties, whereas enhancing expression and deletion of ywtD gene in original strain as well as the effects on the molecular weight and the yield ofγ-PGA in batch culture have not been studied before. Bacillus licheniformis WX-02 is a kind of y-PGA producing bacteria screened by this lab, y-PGA depolymerase of which had been studied.In this study, the ywtD gene expression vectors pHY300-SYA and pHY300-PYA were constructed with SacB promoter and promoter of a-amylase gene from B. licheniformis WX-02, respectively. The recombinants for ywtD gene enhancing expression in B. licheniformis WX-02 was constructed by electrotransformed and designated B. licheniformis SYA2 and PYA3, respectively. The enzyme activity assay showed that the expression of ywtD were enhanced in the recombiants B. licheniformis SYA2 and PYA3. Enzyme activity of B. licheniformis SYA2 and PYA3 determined at OD570 were 0.534±0.002 and 0.560±0.004 respectively, which are higher than 0.503±0.003 of the contrast B. licheniformis PLK. This also suggested that the promoter of P43 is stronger than the promoter of SacB gene induced by sucrose. SDS-PAGE of B. licheniformis SYA2 and PYA3 showed that the YwtD protein was present and active extracellularly in the culture. Both recombinants B. licheniformis SYA2 and PYA3 could reduce the molecular weight of y-PGA from 1000～1200 kDa to about 800 kDa. In addition, B. licheniformis PYA3 could result in a 54% increase of y-PGA production from 13.11 to 20.16 g/L, whereas y-PGA production of B. licheniformis SYA2 reduced to 10.85 g/L. Therefore it is feasible to increase the yield and reduce the molecular weight of y-PGA by enhancing expression of ywtD gene in original strain.Another two integrative vectors pDG780-YA and pNNB194-kanr-YA which are used for ywtD gene deletion was constructed. The mutant with deletion of ywtD gene will be constructed and used for further investigation to elucidate the contribution of ywtD gene to the y-PGA productivity in the next experimentation.