提高重组酵母质粒稳定性和蛋白质产率的培养策略研究

【作者】 夏诏杰；

【导师】 张小里；

【作者基本信息】 西北大学 ， 生物化工， 2002， 硕士

【摘要】 本文以产α—淀粉酶的基因工程酵母菌S.cerevisiae 20B-12(pNA3)为模型体系，在摇瓶培养条件下对外源基因的表达和发酵进行了初步研究，并在2L发酵罐中对发酵工程条件影响质粒稳定性进行了深入的研究，然后对重组酵母发酵动力学进行了数学模拟。 通过继代摇瓶实验发现，在选择性压力下，重组酵母的质粒稳定性在整个培养过程中保持90％以上。当解除选择性压力后，重组酵母的质粒很快出现缺失，但刚开始比率很低，随后质粒的缺失速度加快。重组酵母培养130小时后，质粒几乎100％的缺失。同时我们比较了平板点种法和平板稀释法测定质粒稳定性的方法，两者结果基本一致。 在摇瓶培养中通过均匀实验对培养条件优化，得出在摇瓶中的较佳培养条件：葡萄糖的浓度是34.8g／L，酵母基础氮的浓度是6.6g／L，初始pH是5.8，诱导产酶酵母膏加入时间是当葡萄糖浓度小于0.299g／L。 接着在2L发酵罐中分别考察在诱导阶段温度、溶氧和pH变化对重组酵母S.cerevisiae 20B-12(pNA3)产α-淀粉酶的影响。同时在2L发酵罐中对发酵条件影响质粒稳定性进行了深入的研究，在对表达外源α-淀粉酶的重组酿酒酵母菌株S.cerevisiae 20B-12(pNA3)的培养中发现了营养缺乏的生长静止期质粒稳定性提高的规律。将此应用于诱导分泌表达外源蛋白质的无选择性压力培养阶段，证明脉冲添加酵母膏对稳定质粒同时诱导SUC2启动子控制的外源基因表达是有效的。 结合菌体增殖、质粒稳定及诱导表达的需要，提出了恒流量流加营养物，同时脉冲补加酵母膏的营养物脉冲高密度培养方案，实现了外源蛋白质的效率化生产，最大菌体密度、最大 a－淀粉酶活力分别达到 3 6．4 g／L和207石 U／mL，培养过程质粒保持率稳定于 90％以上。 最后在上述研究的基础上初步建立了重组酵母发酵的动力学模型，并对模型参数进行估计。 由于糖分解酶启动子、营养缺陷型选择性压力在重组酿酒酵母菌株构建中广泛采用，本文提出的培养方案对该类基因工程菌株高密度培养生产外源蛋白质有借鉴意义。更多还原

【Abstract】 In this study the recombinant yeast, S.cerevisiae 20B-12, carrying plasmid pNA3 for production of a -amylase was used as model system to demonstrate the effects of fermentation strategy on enhanced plasmid stability and heterologous protein production. A simple mathematical model was also developed to simulate the fermentation kinetics.Experimental data in continuous propagating flask cultivation showed plasmid stability of recombinant yeast was above 90% under selective pressure, and the plasmid lost immediately when the selective pressure was removed. After 130 hours, no plasmid-carrying cells were detected.The optimization of culture condition was carried out with uniform experimental design method in flask cultivation. The optimal condition was found for flask cultivation with 34.8g/L glucose concentration, 6.6g/L yeast nitrogen base w/o amino acids, initial pH of 5.8 and the favorite feeding time of yeast for inducing enzyme with the glucose concentration below 0.299g/L.The effects of temperature ,dissolved oxygen and pH on the production of a -amylase in the inducing stage were studied in the 2L fermenter. Further studies were carried to demonstrate the feeding strategy for enhanced plasmid stability and protein production. It showed that the plasmid stability increased in the stationary phase with nutrient starvation. With application of this to the inducement and expression of heterologous proteins, the results showed it is effective to enhance the plasmid stability and protein production by pulse-addition of yeast extract.Considering the combination of needs to cell growth, plasmid stability and protein expression, a novel feeding strategy was developed to achieve high plasmid stability and protein production for recombinant yeast fermentation by using constant-addition of SM and pulsing-addition of yeast at the same time. The cell density and the final enzyme activity reached 36.4g/l and 207.6U/mL respectively.A simple mathematical model was also developed to simulate the fermentation kinetics and all parameters in the model were estimated either from direct calculation of the batch fermentation data or model fitting.Since the SUC2 promoter is commonly used in recombinant yeast, the method proposed in this paper could be used for a reference in a scale-up cultivation with a similar recombinant yeast system.更多还原