文献类型：学位论文

中文题名：多拷贝毕赤酵母重组菌表达PIP生理特性和动力学研究

英文题名：Physiological Characterization and Kinetics of Porcine Insulin Precursor Production in Multi-copy Pichia Pastoris

作者：陈丽[1];

机构：[1]华东理工大学;

导师：郭美锦;华东理工大学

授予学位：硕士

语种：中文

中文关键词：重组毕赤酵母;猪胰岛素前体;拷贝数;生理特性;动力学模型;混合补料策略

外文关键词：Recombinant Pichia pastoris; Porcine insulin precursor; Copy number; Physiological characterization; Kinetic models; Mixed feeding strategy

摘要：胰岛素是治疗糖尿病的特效药物之一。本文以本实验室已构建的猪胰岛素前体/(porcine insulin precursor, PIP/)拷贝数分别为1、6、12和18 Mut+型毕赤酵母重组菌为研究对象,考察不同拷贝数毕赤酵母重组菌表达PIP的发酵过程中毕赤酵母生理特性变化和建立发酵动力学模型,同时建立了多拷贝菌株高表达的混合补料策略。 /(1/)通过5L罐补料分批培养,比较拷贝数对毕赤酵母生长、细胞存活率、基因稳定性和OUR等影响,结果表明：在甘油相和诱导初期拷贝数对毕赤酵母的生长无显著影响,但是在诱导24h后高拷贝毕赤酵母菌株/(12拷贝重组菌A2和18拷贝重组菌A3/)比生长速率明显比低拷贝菌株/(1拷贝重组菌G1和6拷贝重组菌G6/)比生长速率慢,A3菌株细胞存活率仅为75/%,且其外源基因丢失率高达19.4/%；四种拷贝数毕赤酵母重组菌中,PIP蛋白产量在12拷贝菌株中最高,为702mg//L。 /(2/)建立发酵过程的菌体生长、产物生成和甲醇累计消耗的动力学模型,确定了模型参数μm、Xm、α、β、κ1、κ2,结果表明发酵的实验值与模型曲线基本吻合,所建立的甲醇补料动力学模型能较好地反映多拷贝毕赤酵母发酵过程,并且模型中与细胞生长相关的产物合成系数α随着拷贝数的增加而增加,说明高拷贝菌株的细胞生长与PIP蛋白表达量具有更紧密的关系。 /(3/)为解决高拷贝菌株生长缓慢问题和提高PIP蛋白表达水平,建立了山梨醇／甲醇混合补料的培养方式,考察了诱导阶段混合补料对不同拷贝数菌株生长和PIP蛋白表达的影响。与仅用甲醇做唯一碳源和能源相比,山梨醇的补入使G1、G6、A2和A3菌株PIP蛋白表达量分别提高1.9/%、42.6/%、34.7/%和80.9/%,这可能由于山梨醇和甲醇混合补料使高拷贝菌株细胞存活率上升,比生长速率增加所致。
Insulin is a potent drug which is currently used in the treatment of diabetes. Recombinant Pichia pastoris Mut+ strains harboring different porcine insulin precursor /(PIP/) gene copy number /(from 1,6,12 to 18/) were genetically constructed in our lab eariler. In this thesis, the effect of PIP gene dosage on physiological characterization of recombinant Pichia pastoris cells was investigated and kinetics of PIP production was established. A mixed feeding strategy for PIP production by multi-copy strains was developed as well. /(1/) The effects of PIP gene copy number on the growth, cell viability, genetic stability and oxygen uptaken rate /(OUR/) of multi-copy P. pastoris cells were studied in 5L fermenter fed-batch culture. The results showed that there were no significant changes in cell growth of multi-copy P. pastoris from the glycerol phase to the early methanol induction phase. However, after 24h of methanol induction, the specific growth rates of both recombinant strain A2 harboring 12-copy PIP gene and strain A3 harboring 18-copy PIP gene remarkably decreased as compared with the low multi-copy strains G1 /(single PIP copy/) and G6 /(six PIP gene copies/). Also, the cell viability of 18-copy strain A3 was only 78/% and its PIP gene loss rate was up to 19.4/% in genetic stability experiments. The maximum PIP production of 702mg//L was achieved by the 12-copy P. pastoris strain A2 grown in fed-batch culture among 4 multiple PIP copies P. pastoris strains tested here. /(2/) Kinetic models of cell growth, PIP formation and total methanol consumption amount in methanol fed-batch culture were proposed and kinetic model parameters ofμm, Xm,α,β,κ1 andκ2 were determined. The experimental data were basically consistent with the model predicted values, indicting that the kinetic models established here, theoretically, provided a reasonable description for multi-copy P. pastoris cells growth in methanol fed-batch culture. With the copy number increasing in recombinant P. pastoris cells, the coefficient of PIP production a was closely associated with the cells growth, indicating that to enhance the specific growth rates of multi-copy strain cells is an effective way to improve PIP production. /(3/) To improve the growth rate of high-pip-copy strains, a mixed feeding strategy with sorbitol and methanol was developed. Compared to methanol as the sole carbon source and energy source, PIP production level has been improved with an increase of 1.9/% for strain G1, 42.6/% for strain G6,34.7/% for strain A2 and 80.9/% for strain A3, respectively, using co-feeds of methanol and sorbitol during the induction phase. It is mainly due to increases in cell viability and in the specific growth rates of high-copy strains grown by co-substrates sorbitol and methanol.