Protein expression and secretion in the yeast Yarrowia lipolytica
Protein expression and secretion in the yeast Yarrowia lipolytica
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DOI：
10.1016/s1567-1356(02)00082-x
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发表时间：
2002-08-01
影响因子：
3.2
通讯作者：
Gaillardin, C
中科院分区：
文献类型：
--
作者：
Nicaud, JM;Madzak, C;Gaillardin, C
已经在酵母解脂耶氏酵母中开发了用于蛋白质表达和分泌的菌株和载体。构建了具有非回复性营养缺陷型标记、蛋白酶编码基因缺失并携带对接平台的宿主菌株。为了驱动转录，使用合成hp 4d或诱导型POX 2启动子。蛋白质分泌由碱性细胞外蛋白酶的靶向序列或细胞外脂肪酶（LIP 2 p）信号序列指导。我们描述了一组载体的基础上，这些启动子，靶向序列和两个URA 3等位基因作为选择标记。野生型UPA 3等位基因ura 3d 1用于单拷贝整合，突变型URA 3等位基因ura 3d 4用于选择多拷贝整合到基因组中。这些载体用于表达Y.解脂菌胞外脂肪酶LIP 2 p和曲霉亮氨酸氨基肽酶II。在hp 4d启动子调控下，单拷贝菌株摇瓶产酶量可达1000 U ml（-1），多拷贝菌株摇瓶产酶量可达2000 U ml（-1），分批产酶量可达11500 U ml（-1），分批补料产酶量可达90500 U ml（-1）。在hp 4d启动子的调控下，单拷贝lapA整合子分批培养的亮氨酸氨基肽酶产量达到320 mU ml（-1），多拷贝lapA整合子分批培养的亮氨酸氨基肽酶产量达到28000 mU ml（-1）。(C)2002年，欧洲微生物学会联合会。由Elsevier Science B. V.出版，版权所有。
Strains and vectors for protein expression and secretion have been developed in the yeast Yarrowia lipolytica. Host strains were constructed with non-reverting auxotrophic markers, deletions of protease-encoding genes, and carrying a docking platform. To drive transcription, either the synthetic hp4d or the inducible POX2 promoter were used. Protein secretion is either directed by the targeting sequence of the alkaline extracellular protease or the extracellular lipase (LIP2p) signal sequence. We describe a set of vectors based on these promoters, targeting sequences and two URA3 alleles as selection markers. The wild-type UPA3 allele, ura3d1, was used for single-copy integration and a mutant URA3 allele, ura3d4, was used to select for multi-copy integration into the genome. These vectors were used to express the Y. lipolytica extracellular lipase LIP2p and the Aspergillus oryzae leucine amino peptidase II. Lipase production under the control of the hp4d promoter by a strain containing a single copy reached 1000 U ml(-1) in shake flasks, while a strain containing multiple integrations reached 2000 U ml(-1) in shake flasks, 11500 U ml(-1) in batch and 90 500 U ml(-1) in fed batch. Leucine amino peptidase production under the control of the hp4d promoter reached 320 mU ml(-1) in batch with a mono-copy lapA integrant and 28 000 mU ml(-1) in fed batch with a multi-copy transformant. (C) 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.