Regulation Mechanism Of Nitrogen On Curdlan Biosynthesis And High Production Strategy In Agrobacterium Sp. ATCC 31749

This dissertation investigated the regulatory mechanism of nitrogen metabolic two-component system NtrB-NtrC on curdlan synthesis and the response of the strain to nitrogen-limited condition based on the knowledge of intracellular carbon and nitrogen metabolism in Agrobacterium sp. ATCC 31749, and by using the theory and methods of metabolic engineering and microbial physiology. The main results are described as follows:(1) The response of Agrobacterium sp. ATCC 31749 to nitrogen limitation from gene transcription and protein expression was analysed by using RT-qPCR and 2-DE methods. Initial results showed that the relative expression of ntrC, ntrB, ntrX, ntrY, glnA, gltB and nifA which were related with nitrogen metabolism and regulation increased significantly. More importantly, the relative expression level of exoC related with carbon flux distribution increased 14-fold in nitrogen-limited condition. In the 2-DE analysis of the cellular total proteins under nitrogen limitation, about 14 proteins expression levels were elevated while 6 of them were decreased. Among them, 4 proteins were successfully identified namely, GroEL, Atu1730, ABC transporter and enoyl-(acyl carrier protein) reductase. The elevated expression of ABC transporter could transport more glucose for curdlan synthesis and decreased expression of enoyl- reductase would channel the flux of UDPG to curdlan production in Agrobacterium sp. ATCC 31749.(2) A ntrC mutant of Agrobacterium sp. ATCC 31749 was constructed and its utiliazation and response of the strain to nitrogen were analysed. Results showed that the consumption rate ofΔntrC on NH₄Cl and KNO₃ decreased, but the mutant could use glutamate and glutamine normally. However, the curdlan synthesis ability was impaired significantly and was lower than 2.0 g/L whatever types of the four nitrogen sources were used. There was a 15 h lag in growth and curdlan production ofΔntrC in batch fermentation with NH₄Cl as nitrogen source. Curdlan production in final medium was 4.8 g/L and was remarkably lower than the production of wild type strain (25.7 g/L). In addition, the cell morphological change rate was also different from that of wild type strain and it was almost in rod type in exponential phase. The nitrogen repression on new biopolymer was released and the mutant could synthesize the new biopolymer at 15 h with sufficient nitrogen in flask fermentation. Comparing to the total protein expression changes at growth phase ofΔntrC and wild type strain, the expression of 43 proteins changed significantly, in which 22 proteins increased and 21 decreased. Four proteins of cobalamin biosynthesis protein, N-acetyl-gamma-glutamyl-phosphate reductase, peptidyl prolyl cis-trans isomerase and nucleoside diphosphate kinase were successfully identified. These results further proved the hypothesis that NtrC was involved in regulating curdlan biosynthesis under nitrogen-limited condition in Agrobacterium sp. ATCC 31749.(3) A ntrB mutant of Agrobacterium sp. ATCC 31749 was also constructed. Results showed that the consumption rate ofΔntrB on NH₄Cl was slower than that of wild type strain, and the mutant could use KNO₃, glutamate and glutamine normally. There was a 5 h lag in growth phase of theΔntrB mutant in batch fermentation with NH₄Cl as nitrogen source, but the yield of biomass to nitrogen (Y_X/N) was identical with that of wild type strain as 1.875. Curdlan synthesis ability ofΔntrB was impaired significantly and only 10.9 g/L was obtained in batch fermentation. The cell morphological change rate was also different from that of wild type strain, but was slower thanΔntrC. The nitrogen repression on the new biopolymer was also released and the mutant could synthesize new biopolymer at nitrogen sufficient condition. By comparing the characteristic ofΔntrC andΔntrB, NtrB is not the unique protein for phosphorylating NtrC, and there might has other proteins, such as protein X of which function was similar to NtrB, but the activation ability of the X protein was lower than NtrB.(4) The two mutants ofΔntrC andΔntrB could synthesize a new biopolymer. The biopolymer has strong water absorption capacity, but could not be dissolved in NaOH, HCl, water and ethyl alcohol. The new biopolymer production ofΔntrC was 6.5 g/L with glutamate as nitrogen source andΔntrB could only produce 2.6 g/L when using KNO₃ as nitrogen source. The infrared spectra analysis showed that structure of the new biopolymer was similar to curdlan sample. It has strong glucosidic bond absorption peak at 1000-1100 cm^-1 and hydroxide radical absorption peak at 3480 cm^-1. But, the difference between the new biopolymer and curdlan was that the new biopolymer does not exhibiteβ-configuration absorption peak at 890 cm^-1. Monosaccharide composition analysis showed that the new biopolymer is composed of glucose, mannose and galactose and other one unknown monosaccharide.(5) Addition of low-polyphosphates with high energy bond for supplying energy was tested to improve curdlan production. In the preliminary experiment two genes encoding the polyphosphate metabolizing enzymes, polyphosphate kinase and exopolyphosphatase were amplified and showed 95% and 86% identical with those in Agrobacterium tumefaciens. C58 and Rhizobium sp. NGR234 by sequence analysis. Three low-polyphosphates (Na₄P₂O₇, Na₅P₃O₁₀ and (NaPO₃)₆) with high energy phosphate bond were employed to substitute for KH₂PO₄-K₂HPO₄ in medium. The curdlan yield was enhanced by 23% and 134% when 0.024 mol/L of Na₅P₃O₁₀ and 0.048 mol/L of (NaPO₃)₆ were added in the medium, respectively. The amount of acetic acid in final fermentation medium decreased 87.5% and 77.7% separately. When CaCO₃ was removed from the culture and the three low-polyphosphates were added, the pH and biomass yield dropped remarkably with little or no curdlan production. The culture with 0.024 mol/L of Na₅P₃O₁₀ and 0.048 mol/L of (NaPO₃)₆ was mixed with KH₂PO₄-K₂HPO₄ and CaCO₃ in the medium showed no effect on curdlan production. However, curdlan yield was improved by 49-60% when CaCO₃ was removed from the medium and KH₂PO₄-K₂HPO₄ were dosed as buffer, and curdlan production reached to 18.4 g/L and 16.9 g/L when 0.024 mol/L and 0.048 mol/L (NaPO₃)₆ were added.