【作者】 唐辉桂；

【导师】 姚斌；

【作者基本信息】 中国农业科学院 ， 生物化学与分子生物学， 2008， 硕士

【摘要】 植酸酶是一种可以水解植酸的磷酸酶类,能够分解饲料中的植酸为动物可利用的无机磷和肌醇,已经在动物营养,人类营养以及酶法合成低磷酸肌醇方面有了很广的应用范围。但目前,提高植酸酶生产菌株的单位表达量,进一步降低其生产成本仍然是最重要的植酸酶产业目标之一。本研究以目前植酸酶表达量最高的实际生产菌株74#为材料,通过共表达二硫键异构酶、透明颤菌血红蛋白,增加基因拷贝数等方法,以期进一步提高其表达量,降低生产成本。本研究也对毕赤酵母表面展示植酸酶进行了尝试。根据已发表的毕赤酵母来源二硫键异构酶(PDI)基因序列,克隆获得PDI编码基因,并构建了含有PDI编码基因的毕赤酵母胞内表达载体pPICZA-pdi,以植酸酶生产菌株74#为受体进行了转化。从600个转化子中筛选到3个酶活性更高的重组子,其中酶活性最高的49#菌株其活性是原有菌株74#酶活性的120%,在5L发酵罐中表达活性达到16,582 U/mL。在此基础上,用经密码子优化的大肠杆菌植酸酶基因appA-m,构建了表达载体pPIC9K-appA-m。以共表达PDI蛋白的植酸酶菌株49#为受体,筛选重组子后得到多拷贝的植酸酶菌株79#,酶活性达到17,434 U/mL,与共表达PDI蛋白的49#菌株相比,提高了10%;与生产菌株74#相比,提高了34%。根据已发表的树干毕赤酵母基因组序列克隆了低氧启动子PsADH2 promoter序列,并根据已发表的透明颤菌血红蛋白序列,按照毕赤酵母密码子偏爱性对血红蛋白基因进行了密码子改造和合成。构建了表达载体pPIC9K-PsADH2-vgb-m,以植酸酶生产菌株74#为受体进行了转化。对重组子进行筛选得到低氧条件下植酸酶活性最高的转化子15#,利用摇瓶培养,其在低氧条件下的酶活性是74#菌株酶活性的4倍,达到249 U/mL。另外,本研究对表面展示植酸酶进行了尝试。通过已发表的啤酒酵母基因组序列克隆了α凝集素蛋白AGα1编码基因,构建了表达载体pPIC9K-appA-m-AGα1,转化毕赤酵母,对重组子进行筛选,得到了在酵母细胞表面有植酸酶活性的菌株。更多还原

【Abstract】 Phytase (myo-inositol hexakisphosphate phosphohydrolases) can hydrolyze phytate to lower myo- inositol phosphates and inorganic phosphate and has extensive applications in animal nutrition, human nutrition and synthesis of lower inositol phosphates. One of the main objectives of phytase industrial production is to further improve the protein expression level of the phytase production strain and reduce its production costs.Using the commercial phytase strain #74 as the host strain, which has the highest expression level at present, through the coexpression of protein disulfide isomerase and Vitreoscilla hemoglobin(VHb), and increasing the appA-m gene copy number, we aimed at further improving the phytase production level and decreasing the costs. Besides, the cell surface display of the phytase on P. pastoris was also tried.According to the reported gene sequence (GenBank accession number AJ302014) of disulfide isomerase (PDI) from Pichia pastoris, the PDI encoding gene was cloned and then ligated into the vector pPICZA. Plasmid pPICZA-pdi was then transformed into the competent cell of P. pastoris #74. Among the 600 positive clones, P. pastoris #49 had the highest phytase activity of 16,582 U/mL in a 5 L fermenter, 20% more than that of P. pastoris #74. Western blot result revealed that the PDI was functionally expressed in P. pastoris #49 with the molecular mass of about 65 kD, resulting in the improvement of phytase production. Using the E. coli phytase gene appA-m which has been modified according to the codon bias of P. pastoris, the expression vector pPIC9K-appA-m was constructed and transformed into P. pastoris #49 for multicopy integration. Among the 300 clones, P. pastoris #79 showed the highest phytase activity of 17,434 U/mL, 10% higher than that of P. pastoris #49 and 34% higher than that of P. pastoris #74.According to the reported sequence (EMBL accession numbers Y13397 and AF008245), the ADH2 promoter of 600 bp was obtained from Pichia stipitis. Moreover, based on the Vitreoscilla hemoglobin sequence (GenBank accession number M27601), the vgb gene was modified and synthesized according to the codon bias of P. pastoris, without changing the amino acid sequence of VHb. The PsADH2- promoter and the vgb-m gene were fused through overlap PCR. The P. pastoris expression vector pPIC9K-PsADH2-vgb-m was then constructed and transformed into P. pastoris #74. Under oxygen limitation, P. pastoris #15 showed the highest phytase activity of 249 U/mL, three times higher than that of P. pastoris #74.Besides, we also tried the cell surface display of E. coli phytase. Based on the published sequence ofα-agglutinin (Genbank accession number X16861) from Saccharomyces cereviase, AGα1 encoding gene was obtained by PCR and expression vector pPIC9K-appA-m-AGα1 was constructed, and then transformed into P. pastoris GS115. Phytase activity was detected on the yeast cell surface.更多还原