磷酸盐浓度对铜绿假单胞菌PAO1产鼠李糖脂及其基因表达的影响

发布时间：2018-03-23 03:14

本文选题：铜绿假单胞菌　切入点：鼠李糖脂　出处：《西北大学》2017年硕士论文　论文类型：学位论文

【摘要】：鼠李糖脂是近年来受到广泛关注的生物表面活性剂之一,因应用范围广以及环境友好等特点,使其成为潜在的合成表面活性剂的替代品。然而,鼠李糖脂的生产存在产量低、规模小以及成本高等问题,大大限制了其在工业规模上的使用。铜绿假单胞菌(Pseudomonas aeruginosa)是发酵生产鼠李糖脂最理想的菌株之一,但其鼠李糖脂的生物合成受到环境因子、营养因素以及复杂基因调控的影响,其中基因表达调控QS系统(rhl系统、las系统和pqs系统)的作用又与环境因子以及营养因素密切相关。本研究以铜绿假单胞菌(Paerwginosa)PAO1为对象,采用Plackett-Burman(PB)设计和响应曲面方法(RSM)对其产鼠李糖脂的发酵条件进行多因素优化。PB试验设计显示磷酸盐浓度、pH值和C/N比3个因素对鼠李糖脂的产量具有显著影响,其中最显著的影响因素是磷酸盐浓度;通过RSM对3个显著因素的最佳水平范围进行分析,结果表明当磷酸盐浓度为1.71 g/L、pH值为6.0、C/NL比为15.5时,菌株PAO1合成鼠李糖脂的产量可高达20.72 g/L,比优化前鼠李糖脂产量12.63 g/L提高了64.05%,很接近理论预测值21.35 g/L。在此基础上,选取最显著影响因素磷酸盐浓度,研究不同磷酸盐浓度对菌株PAO1合成鼠李糖脂的影响,结果发现当磷酸盐浓度为0.01-0.5 g/L(即低磷浓度)时,其鼠李糖脂的产量比最佳水平磷酸盐浓度时的产量高;尤其当磷酸盐浓度为0.5 g/L时,鼠李糖脂产量最高达到24.93 g/L。但是,当培养基中不含磷酸盐时,鼠李糖脂的产量仅有16.86 g/L。血平板实验显示在磷酸盐浓度为0.5 g/L时溶血圈最大,与鼠李糖脂的产量结果一致;SDS-PAGE凝胶电泳分析发现菌株PA01在不同磷酸盐浓度下有相应的差异蛋白表达。采用qPCR的方法,对不同磷酸盐浓度下rhl系统、las系统以及双组份调控系统相关基因的相对表达量进行测定。结果显示:rhlI、rhlR、lasI、lasR、rsaL、phoB、phoR基因的相对表达量均发生明显的变化。由此可见,不同磷酸盐浓度对铜绿假单胞菌合成鼠李糖脂及其基因表达调控有影响。为了进一步探究铜绿假单胞菌在不同磷酸盐浓度下对鼠李糖脂合成的影响,构建了 rhl系统突变菌(△rlI、△rhlR)和las系统突变菌(△lasI、△lasR、△rsaL),分别对其在不同磷酸盐浓度下鼠李糖脂的产量及其相关基因的表达调控进行研究。结果表明:在低磷条件下,△rhlI和△rhlR的鼠李糖脂产量(18.35 g/L和16.04 g/L)相比PA01的产量(24.93g/L)分别下降了 26.39%和35.66%,△lasI和△lassR的鼠李糖脂产量变化不明显,尤其是AlasI在此条件下的合成量(23.3 g/L)与PA01的产量几乎一致,而△rsaL的鼠李糖脂产量(28.36g/L)比PA01的产量提高了 13.88%。qPCR结果显示:rhl系统突变菌中rhlA、rhlB基因的相对表达量较低,但△lasI和△lasR中rhlA、rhlB基因的相对表达并未受到太大影响,而ArsaL中rhlA、rhlB基因的相对表达升高;rhl系统突变菌和△rsaL中phoB、phoR基因的相对表达量下降,AlasI和△lasR中相对表达量无明显变化。在无磷条件下,△rhlR和△lasR的鼠李糖脂产量(12.86g/L和13.02 g/L)相比野生菌 PAO1 的产量(16.86 g/L)分别下降了 15.79%和 22.78%,ArhlI和△lasI在此条件下的鼠李糖脂合成量与野生菌的产量一致,而△rsaL的产量(20.12 g/L)比PAO1的产量提高了 19.34%。qPCR结果显示,△tasR和△rhlR中基因rhlA、rhlB的相对表达量有明显的下调,而△rsaL中表达量明显上调;phoB、phoR基因的相对表达量在各菌株中几乎不变。由此可见,低磷条件下,rhl系统在鼠李糖脂合成和调控中起关键作用,双组份调控系统也参与了鼠李糖脂的调控;无磷条件下,rhl系统中的rhlR基因和las系统中的lasR基因在鼠李糖脂生物合成及基因表达调控中起主要作用,而双组份调控系统不参与调控鼠李糖脂的合成;rsaL基因作为las系统的抑制子,会抑制鼠李糖脂的合成。因此,鼠李糖脂的合成及其基因表达调控受到了营养因素的影响;在不同磷酸盐条件下,铜绿假单胞菌适应环境的应答机制有所差异,能直接或间接影响鼠李糖脂的合成。
[Abstract]:Rhamnolipid is one of attention in recent years because of the characteristics of biosurfactant, wide application range and environment friendly, make it become the synthetic surfactant potential alternatives. However, rhamnolipid production has low yield, small size and high cost, which greatly limits its use in industrial scale.. Pseudomonas aeruginosa (Pseudomonas aeruginosa) is one of the most ideal strains fermentation production of rhamnolipid biosynthesis, but its rhamnolipid by environmental factors, nutritional factors and complicated gene regulation, including gene expression and regulation of QS system (RHL system, Las system and PQS system) function is closely related to nutrition and environmental factors. In this study, Pseudomonas aeruginosa (Paerwginosa) PAO1 as the object, using Plackett-Burman (PB) design and response surface method (RSM) on the production of rhamnolipid The fermentation conditions were optimized.PB multi factor experimental design showed that phosphate concentration has significant effect, pH value and C/N ratio of 3 factors on rhamnolipid production, the most influential factor is the concentration of phosphate; through the RSM of the 3 significant factors of the optimal level of analysis, the results show that when the phosphate concentration was 1.71 g/L, pH value was 6, C/NL was 15.5, the strain PAO1 synthesis of rhamnolipid yield can be as high as 20.72 g/L, 64.05% higher than before the rhamnolipid production optimization of 12.63 g/L, very close to the theoretical prediction value of 21.35 g/L. on the basis of this, select the most significant factors affecting phosphate concentration, influence of phosphate concentration on bacteria PAO1 rhamnolipid synthesis, the results showed that when the concentration of phosphate was 0.01-0.5 g/L (i.e. low phosphorus concentration), the rhamnolipid yield than the optimal level of phosphate concentration when the high yield; especially when phosphate The concentration of 0.5 g/L, the highest rhamnolipid yield reached 24.93 g/L., but when the culture medium does not contain phosphate, rhamnolipid yield only 16.86 g/L. blood agar experiments showed that in phosphate concentration was 0.5 g/L hemolysis of the largest circle and rhamnolipid yield consistent results; analysis showed that strain PA01 had the corresponding differences in protein expression at different concentration of phosphate by SDS-PAGE gel electrophoresis. By using qPCR method of RHL system with different concentration of phosphate, Las related gene and two-component regulatory system expression were determined. The results show: rhlI, rhlR, lasI, lasR, rsaL, phoB, the relative expression of phoR gene were significantly change. Thus, different concentration of phosphate has effects on Pseudomonas aeruginosa rhamnolipid synthesis and gene expression. In order to further explore the Pseudomonas aeruginosa in acid salt under different phosphorus concentration The effects of rhamnolipid synthesis, constructs the RHL system mutants (rlI, rhlR) and Las system mutant (delta lasI, Delta lasR, Delta rsaL), respectively. The expression of rhamnolipid in different phosphate concentrations and lipid yield and its related genes were studied. The results showed that under low phosphorus under the condition of delta rhlI and delta rhlR rhamnolipid yield (18.35 g/L and 16.04 g/L) compared to the yield of PA01 (24.93g/L) were decreased by 26.39% and 35.66%, rhamnolipid production changes of delta lasI and delta lassR is not obvious, especially the amount of AlasI synthesized under this condition (23.3 g/L) is almost the same with PA01 the yield, and the yield of rhamnolipid (rsaL 28.36g/L) than the PA01 yield increased by 13.88%.qPCR showed that RHL mutant rhlA, the relative expression of rhlB gene was low, but the rhlA Delta lasI and delta lasR, the expression of rhlB gene is not influenced, ArsaL rhlA, the relative expression of rhlB gene increased; RHL mutation system phoB bacteria and rsaL, the relative expression of phoR gene decreased the amount of AlasI and lasR in relative expression did not change significantly. In the absence of phosphorus conditions, Delta rhlR and delta lasR rhamnolipid yield (12.86g/L and 13.02 g/L) compared wild mushroom yield of PAO1 (16.86 g/L) were decreased by 15.79% and 22.78%, uniform production of rhamnolipid synthesis of ArhlI and lasI under this condition with the wild strain, and the yield of rsaL (20.12 g/L) than the PAO1 yield increased by 19.34%.qPCR showed that rhlA gene of delta tasR and delta rhlR, rhlB the relative expression was down regulated, while rsaL expression was significantly upregulated; phoB, the relative expression of phoR gene is almost unchanged in all strains. Thus, low phosphorus conditions, RHL system play a key role in rhamnolipid synthesis and regulation, two-component The regulation system is also involved in the regulation of rhamnolipid; no phosphorus conditions, lasR gene rhlR and LAS in RHL system of expression plays an important role in regulation of rhamnolipid biosynthesis and gene synthesis, and two-component regulation system is not involved in the regulation of rhamnolipid; rsaL gene as a suppressor of Las system. Inhibition of rhamnolipid synthesis. Therefore, synthesis and gene expression regulation of rhamnolipid was influenced by nutritional factors; in different phosphate conditions, Pseudomonas aeruginosa adaptive response mechanism of environment is different, can directly or indirectly affect the synthesis of rhamnolipid.

【学位授予单位】：西北大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q78;Q939.9

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