Establishment of an overall transformation system for an oil-producing filamentous fungus, Mortierella alpina 1S-4
Establishment of an overall transformation system for an oil-producing filamentous fungus, Mortierella alpina 1S-4
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DOI：
10.1007/s00253-004-1622-6
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发表时间：
2004-09-01
影响因子：
5
通讯作者：
Shimizu, S
中科院分区：
文献类型：
--
作者：
Takeno, S;Sakuradani, E;Shimizu, S
高山被孢霉1 S-4是一株高产油脂的工业菌株。为了确定其生理特性并阐明多不饱和脂肪酸的生物合成途径，使用其衍生物，即，缺乏乳清酸磷酸核糖转移酶（OPRTase，EC. 2.4.2.10）活性的ura 5突变体。用含有作为标记的同源ura 5基因的载体进行转化，成功地进行了使用微粒轰击，其他方法经常用于转化，如原生质体，乙酸锂，或电穿孔方法，不能得到令人满意的结果。结果，获得两种类型的转化体：一些稳定的转化体过表达ura 5基因，和许多不稳定的转化体显示OPRTase活性与野生型菌株相当。定量PCR结果表明，无论培养条件如何，稳定的转化子都能保留来自转化载体的ura 5基因。另一方面，不稳定的转化体在含尿嘧啶的条件下容易丢失标记基因，正如预期的那样。本文报道了该菌的一套完整的转化体系的建立，并提出了如何筛选转化子作为实验菌株和工业菌株的建议。
Oil-producing fungus Mortierella alpina 1S-4 is an industrial strain. To determine its physiological properties and to clarify the biosynthetic pathways for polyunsaturated fatty acids, a transformation system for this fungus was established using a derivative of it, i.e., a ura5 mutant lacking orotate phosphoribosyl transferase (OPRTase, EC.2.4.2.10) activity. Transformation with a vector containing the homologous ura5 gene as a marker was successfully performed using microprojectile bombardment, other methods frequently used for transformation, such as the protoplasting, lithium acetate, or electroporation methods, not giving satisfactory results. As a result, two types of transformants were obtained: a few stable transformants overexpressing the ura5 gene, and many unstable transformants showing OPRTase activity comparable to that of the wild-type strain. The results of quantitative PCR indicated that the stable transformants could retain the ura5 genes originating from the transformation vector regardless of the culture conditions. On the other hand, unstable transformants easily lost the marker gene under uracil-containing conditions, as expected. In this paper, we report that an overall transformation system for this fungus was successfully established, and propose how to select useful transformants as experimental and industrial strains.