【作者】 于丽珺；

【导师】 詹晓北；

【作者基本信息】 江南大学 ， 发酵工程， 2011， 博士

【摘要】 本文以一株能大量合成胞外多糖热凝胶的土壤杆菌31749 （Agrobacterium sp. ATCC 31749）为研究对象,在初步了解土壤杆菌31749胞内碳源及氮源代谢的基础上,运用代谢工程和微生物生理学的理论与方法,就氮代谢双组分系统NtrB-NtrC调控土壤杆菌31749在氮源限制条件下合成胞外多糖的机理展开研究。主要研究结果如下:（1）采用RT-qPCR和2-DE技术研究了土壤杆菌31749氮源代谢调控系统和菌体总蛋白对氮源限制环境的应答变化。结果表明,氮源限制条件可以显著提高氮源代谢基因glnA、gltB、nifA及其相关调控基因ntrC、ntrB、ntrX、ntrY的相对转录水平,并将碳代谢流分配关键基因exoC的相对转录水平提高了14倍。蛋白质二维电泳分析发现,土壤杆菌31749在应答环境氮源含量变化时,14个蛋白质表达量显著提高,6个蛋白质表达量下调。这20个蛋白质中有4个被成功鉴定,分别为分子伴侣GroEL、未知蛋白Atu1730、ABC转运蛋白和烯酰ACP还原酶。ABC转运蛋白表达水平的提高满足菌体大量转移糖类物质的需求,烯酰ACP还原酶表达量的下调使菌体细胞壁合成受阻,而用于细胞壁合成的UDPG被主要用于热凝胶的合成。（2）利用同源重组原理构建土壤杆菌31749的ntrC突变株ΔntrC,分析ΔntrC对氮源的应答和利用情况。结果表明:ΔntrC对培养基中NH₄Cl和硝酸钾的利用速度显著减慢,但能正常利用谷氨酸和谷氨酰胺。无论ΔntrC利用这4种氮源中的任何一种,热凝胶合成量均小于2.0 g/L。在以NH₄Cl为氮源的分批发酵过程中,ΔntrC的热凝胶合成时间相对野生菌延迟15 h,最终发酵液热凝胶含量为4.8 g/L,显著的低于野生菌热凝胶产量（25.7 g/L）。另外,ΔntrC在菌体形态变化速度上显著不同于野生菌,其形态在对数生长期几乎全是杆状。通过分析ΔntrC和野生菌在生长期的总蛋白表达差异,发现43个蛋白质表达量发生显著变化,22个表达上调,21个表达下调。43个蛋白质中有4个表达量变化显著的被成功鉴定,分别为钴胺素生物合成蛋白、肽基脯氨酰顺反异构酶、核苷二磷酸激酶和N-乙酰-γ-谷氨酰磷酸脱氢酶。上述结果也证实最初假设,即NtrC参与调控热凝胶的合成。（3）利用同源重组原理进一步构建土壤杆菌31749的ntrB突变株ΔntrB,并分析ΔntrB对环境氮源应答和利用情况。结果表明:ΔntrB对NH₄Cl的利用速度显著减慢,但能正常利用硝酸钾、谷氨酸和谷氨酰胺。以NH₄Cl为氮源的分批发酵过程中,ΔntrB氮源消耗时间延长5 h,但生物量对氮源得率(Y_X/N)相比野生菌基本不变,仍维持在1.875 g/g的水平;而突变株ΔntrB合成热凝胶的能力显著削弱,热凝胶产量仅为10.9 g/L。ΔntrB的菌体形态变化速度也较野生菌加快,但是变化速度没有ΔntrC快。通过比较ΔntrC和ΔntrB特性发现,NtrB并不是唯一能对NtrC进行磷酸化的蛋白,可能存在其他的此类蛋白X,但是X蛋白对NtrC的磷酸化能力比NtrB低。（4）ΔntrC和ΔntrB都开启一种新聚合物的合成。ΔntrC利用谷氨酸为氮源时所产新聚合物产量最高,达到6.5 g/L;ΔntrB以KNO₃为氮源时新聚合物产量只有2.6 g/L。ΔntrC和ΔntrB解除了氮源对新胞外聚合物合成的限制作用,在氮源充足条件下,二者都能合成新的胞外聚合物。新聚合物具有极强的吸水性,但不溶于水,也不溶解于NaOH、HCl和无水乙醇。红外光谱扫描结果发现,新聚合物的吸收峰与热凝胶标样的吸收峰大部分一致。样品在1000-1100 cm^-1和3480 cm^-1分别有很强的糖苷键和羟基的特征吸收;但是新聚合物不同于热凝胶是在890 cm^-1处没有明显的β–构型的特征吸收峰。单糖组成分析发现,新聚合物由葡萄糖、甘露糖、半乳糖和其余一种未知的单糖组成。（5）通过添加高能化合物为土壤杆菌31749合成热凝胶提供能量以提高胞外多糖产量。首先确定了土壤杆菌31749染色体基因组上存在多聚磷酸激酶和外切聚磷酸酶,其中多聚磷酸激酶编码基因ppk序列与Agrobacterium tumefaciens. C58的同源基因有95%的一致性,而外切聚磷酸激酶编码基因ppx与Rhizobium sp. NGR234染色体上此基因有86%的一致性。利用3种具有不同高能磷酸键的低聚磷酸盐Na₄P₂O₇、Na₅P₃O₁₀和（NaPO₃）₆取代培养基中的KH₂PO₄-K₂HPO₄,将它们作为高能磷酸键供体和磷元素营养添加到热凝胶发酵体系中。结果表明:在培养基中分别添加0.024 mol/L的Na₅P₃O₁₀和0.048 mol/L的（NaPO₃）₆,对应的热凝胶产量较对照分别提高了23%和134%,而副产物乙酸较对照分别降低87.5%和77.7%,表明低聚磷酸盐的添加促进了细胞代谢过程的能量供给,在缓减副产物积累的同时又强化了热凝胶的合成。当除去上述发酵体系中的CaCO₃,添加上述3种低聚磷酸盐时,生物量显著降低,几乎不合成热凝胶,发酵液pH最低降到2.1。当同时以CaCO₃和KH₂PO₄-K₂HPO₄作为缓冲物质,分别添加0.024 mol/L的Na₅P₃O₁₀和0.048 mol/L的（NaPO₃）₆发酵时,热凝胶产量变化不显著。但是,当发酵液不存在CaCO₃,只有KH₂PO₄-K₂HPO₄作为缓冲物质时,添加0.024 mol/L和0.048 mol/L的（NaPO₃）₆将使热凝胶分别达到18.4 g/L和16.9 g/L,较对照分别提高60.4%和49.4%。更多还原

【Abstract】 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.更多还原