Studies On Efficient Bioproductions Of Poly(γ-glutamic Acid) And Poly(β-L-malic Acid)

As two novel biodegradable biomaterials, Poly-γ-glutamatic acid (γ-PGA) and poly (β-L-malic acid)(PMLA) have great potential applications in medicine and a wide range of other areas because of their excellent features. Microbial syntheses of the two biopolymers have been studied systematically in the present work.Firstly, a glutamic acid-independent strain was screened out from sauce samples to biosynthesize γ-PGA without extrogeneous glutamic acid addition. According to the VITEK system identification and16srDNA sequence analysis, this new isolated strain was nominated as Bacillus subtilis C10. The metabolic characteristics and culture conditions of C10were investigated for enhanced γ-PGA production. The results showed that extrogeneous citric acid could be uasd as precuisor for γ-PGA synthesis, and one fed-batch strategy was examined to further improve the productivity. The mixture solution containing citric acid and ammonium chloride was fed in the later culture stage of fermentation, which resulted in high γ-PGA(38.2g/L) production. The factors influencing the endogenous glutamic acid supply and the biosynthesis of γ-PGA were also investigated in this strain, especially the effects of five different organic acids as possible precursors on the production of γ-PGA. To understand the possible mechanism for the improved γ-PGA biosynthesis by the tested organic acids, the activities of four key intracellular enzymes were measured. The result indicated that the increased bio activity of pyruvate dehydrogenase by oxalic acid was important for de novo sythesis of glutamic acid from glucose. According to metabolic pathway analyses, citric acid directly entered into the biosynthetic pathway of γ-PGA through the TCA cycle as indirect substrate of glutamate synthetase, while oxalic acid did not enter the biosynthetic pathway of γ-PGA. Metabolic flux analyses of γ-PGA synthesis in strain C10were carried out to explain the different effects caused by the addition of these organic acids. The feeding of citric acid reduced the flux entering the EMP pathway and increased the flux to γ-PGA synthesis pathway in α-ketoglutaric acid node. With the addition of oxalic acid, more carbon flux entered the TCA cycle in this strain. In order to improve the production of γ-PGA, the α-ketoglutaric acid dehydrogenase activity should be weakened in this glutamic acid-independent γ-PGA producer.Secondly, a new strain with the distinguished high productivity of PMLA was isolated from fresh plant samples. According to morphological characteristics and phylogenetic analyses of the internal transcribed spacer sequences, one PMLA-producing strain (ZD-3d) was characterized as the candidate of Aureobasidium pullulans. As expected, PMLA can be hydrolyszed into malic acid, maleic acid and fumaric acid, and the corresponding kinetics of PMLA acid hydrolysis was modeled to simulate the whole degradation process. Further culture condition optimization brought about the highest PMLA concentration (62.27g/L) in the shake flask scale. In addition, the contribution of the carbon flux to exopolysaccharide (EPS) and PMLA could be regulated by the addition of CaCO3in the medium. This high-level fermentation process was further scaled up in the10L bench-top fermentor with a high PMLA concentration (57.2g/L), productivity (0.35g/L/h) and yield (0.47g/g glucose).The present work made some deep insights into the biosynthesis and regulation of γ-PGA and PMLA in their corresponding strains and promises some novel strategies for industrial production of these two important biopolymers in the future.