基于基因的重新设计与高通量筛选策略选育解脂耶氏酵母脂肪酶高产菌株  

De Novo Design and High-throughput Screening Strategy Achieved Over Expression of Yarrowia lipolytica Lipase YLL in Pichia patoris

作　　者：杨江科[1] 毛玲[1] 周文静[1] 陈江山[1] 胡臣[1] 

机构地区：[1]武汉轻工大学生物与制药工程学院,武汉430023

出　　处：《中国生物工程杂志》2014年第8期54-60,共7页China Biotechnology

基　　金：国家"863"计划(2013AA102805-4);教育部科学技术研究重点项目(212118);湖北省教育厅科学技术研究计划(D20131703)资助项目

摘　　要：脂肪酶(EC 3.1.1.3)是应用广泛的工业用酶。高效的脂肪酶产生菌是脂肪酶工业生产和应用的前提。通过基因的重新设计与合成技术优化了解脂耶氏酵母(Yarrowia lipolytica)脂肪酶YLL的密码子,并实现了其在毕赤酵母(Pichia pastoris)中的高效表达;通过高通量筛选策略获得了更高效的脂肪酶基因工程菌菌株SILVER。在14 L发酵罐条件下,菌株SILVER酶活达40 500U/ml、蛋白质含量达2.52 g/L发酵液,为该类脂肪酶的产业化奠定了基础。Lipase（ EC 3. 1. 1. 3） is a important biocatalyzer used in a variety of industrial fields. High-level lipase producing strains is the prerequisite for industrial bioapplication of lipase. De novo design,gene synthesis and high-throughput screening strategies were used to realize the overexpression of Yarrowia lipolytica lipase YLL in Pichia pastoris. By de novo design and gene synthesis of YLL lipase gene,the expression level of synthesized lipase gene was improved 1. 26 fold than the original YLL gene. After that,a high throughput screening strategy including olive oil Rhodamine B plates phenotype check,96 well plates and flask fermentation was used to acquire the over expression Pichia recombinant SILVER. In the 14 L fermentor,Pichia recombinant strain SILVER achieved the highest activity of 40 500 U /ml broths,and the protein content reached 2. 52 g /L broths,which set the foundation for industrial application of YLL lipase.

关 键 词：脂肪酶 基因设计 高通量筛选 高效表达 发酵 

分 类 号：Q812[生物学—生物工程]