Department of Biotechnology, Yuanpei University, Hsin Chu, Taiwan.
N Biotechnol. 2010 Sep 30;27(4):277-82. doi: 10.1016/j.nbt.2010.02.006. Epub 2010 Feb 25.
The hybrid promoter (hp4d) expression cassette, one of the efficient tools of Yarrowia lipolytica expression system, has been applied to produce or secrete a variety of recombinant proteins. This cassette directs a strong gene expression, because the hp4d promoter exhibits high level quasi-constitutive activity. The objective of this study is to test whether two expression cassettes inserted into a vector could function efficiently and simultaneously. Taking advantage of the well-known biosynthesis pathway of gamma-linolenic acid (GLA), we examined the performance of Y. lipolytica, transformed with two expression cassettes containing previously cloned Delta12-desaturase and Delta6-desaturase genes, by monitoring fatty acid composition of cellular lipids. Our results confirmed that each individual desaturase gene was expressed efficiently by the expression cassette. When two cassettes with respective desaturase genes, carried on the same vector, were integrated into yeast genome, a significant level of GLA was synthesized from endogenous linoleic acid (LA) and oleic acid (OA). Besides, both expression cassettes functioned effectively without influence from each other. These findings indicated that co-expression of two desaturase genes by this dual cassette vector was effective and simultaneous. Results from the present study provide an alternative approach for both the production of several proteins at the same time, and the development of single cell oil containing high-valued polyunsaturated fatty acids (PUFAs).
杂交启动子（hp4d）表达盒是假丝酵母表达系统的有效工具之一，已被用于生产或分泌各种重组蛋白。该表达盒可实现高效的基因表达，因为 hp4d 启动子具有高水平的准组成型活性。本研究的目的是测试两个插入载体的表达盒是否能同时有效地发挥作用。利用γ-亚麻酸（GLA）的著名生物合成途径，我们通过监测细胞脂质的脂肪酸组成，检测了转化为含有先前克隆的Δ12-去饱和酶和Δ6-去饱和酶基因的两个表达盒的假丝酵母的性能。我们的结果证实，每个单独的去饱和酶基因都可以通过表达盒高效表达。当带有各自去饱和酶基因的两个载有各自去饱和酶基因的表达盒被整合到酵母基因组中时，从内源性亚油酸（LA）和油酸（OA）中合成了大量的 GLA。此外，两个表达盒在不受彼此影响的情况下有效地发挥作用。这些发现表明，通过这种双盒载体共表达两个去饱和酶基因是有效和同时的。本研究结果为同时生产多种蛋白质和开发富含高价值多不饱和脂肪酸（PUFA）的单细胞油提供了一种替代方法。
