| Poly-gamma-glutamic acid (y-PGA) is a polypeptide which can be produced by microbial fermentation. It is widely used in the fields of foods, cosmetics, Pharmaceuticals and water treatment due to its outstanding qualities such as biodegradability, film-reforming property, moisture-retention property and edibility. In the previous study, we took a glutamate-dependent y-PGA producing strain, Bacillus subtilis HSF1410, as study object and found that the addition of CaCl2 could improve the yield of y-PGA and decrease the viscosity of broth as well. During the study into the mechanism of y-PGA synthesis, it was showed that calcium could improve the expression of gdh at transcriptional level and thus increase the activity of glutamate dehydrogenase (GDH) at the a-ketoglutaric acid joint. In addition, the activity of OGDH increased with the concentration of calciumTo analyze the a-ketoglutaric acid joint thoroughly and dig into the regulation mechanism of calcium on y-PGA synthesis, we studied the effect of calcium on activity of a-ketoglutaric acid dehydrogenase (OGDH) in vivo and in vitro, as well as expression abundances of ogdh and pgsB. In addition, the variation trend of the field of y-PGA, the consumption of NH4Cl and biomass were determined.To better understand the regulation mechanism on a molecular level, the complete genome sequences were determined and the annotation of non-coding RNA, protein function and etc. were conducted. The phylogenetic trees of y-PGA producing strains were built based on pgsB and 16s rRNA to analyze the classification of glutamate-dependent and-independent strains. Our study results are as follows:(1) The regulation mechanism of calcium on OGDH was studied from the point of in vivo and in vitro. The results showed that calcium had no direct effect on OGDH in vitro; the transcription ratios of ogdh were determined after HSF1410 being cultivated with the addition of calcium, results showed that when the concentration of calcium increased from 0 to 0.2%o, the transcription ratio of ogdh and enzyme activity of OGDH increased by 3.518 times and 2.2 times respectively. Therefore, it can be concluded that the transcription level of odgh and activity of OGDH increased with the concentration of calcium, thus the metabolic flux in TCA circle increased and more carbon and nitrogen sources were used to synthesize glutamate, improving y-PGA synthesis.(2) The regulation mechanism of calcium on PgsB was studied from the level of transcription. When the concentration of calcium increased from 0 to 0.2%o, the transcription ratios of pgsB increased by 0.76 times. PgsB catalyzes the extension of nitrogen bone in y-PGA, therefore improvement of transcription ratios of pgsB could boost y-PGA production.(3) The effect of calcium on biomass growth of B. subtilis HSF1410, field of y-PGA and consumption of NH4CI was studied. We found that biomass of HSF1410 declined with the concentration of calcium, indicating that calcium inhibited the growth of bacterium to a certain degree. The field of y-PGA and the consumption of NH4CI experienced an upward trend and then declined, and reached a maximum of 8.55 g/1 and 0.698 g/1 respectively when 0.1%o of CaCl2 was added in medium. The maximum conversion rate of glutamate was 0.428. In conclusion, the optimal concentration of CaCl2 is 0.1‰ (w/v).(4) The complete genome sequences were determined using high-throughput sequencing (HTS) technology. The genome of B. subtilis HSF1410, with a GC content of 43.26%, is composed of a 4.18Mb chromosome. The gene sequences were assembled using Glimmer3.0 and annotation information of the corresponding proteins were acquired by assignment using the BLAST software running against a composite protein databank compound of KEGG、COG、SwissProt.(5) Pylogenetic analysis based on pgsBCA and 16s rRNA showed that these two types of y-PGA-producing strains are distinguishable based on 16S rRNA and pgsBCA phylogenies as well, which is consistent with the different requirements of fermentation medium of these strains.In conclusion, this research found that the activities of OGDH and PgsB are regulated by Ca2+ at transcriptional level and have a significant effect on the field of y-PGA in turn. These experimental data could point the way for further research into the regulation mechanism of y-PGA synthesis. In addition, we provided the information of complete genome sequence of HSF1410, laying a theoretical foundation for establishing molecular operation system and studying on molecular mechanism. |
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