Heterologous expression of glucose oxidase in the yeast Kluyveromyces marxianus.
Heterologous expression of glucose oxidase in the yeast Kluyveromyces marxianus.
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DOI：
10.1186/1475-2859-9-4
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发表时间：
2010-01-21
影响因子：
6.4
通讯作者：
Gombert AK
中科院分区：
文献类型：
--
作者：
Rocha SN;Abrahão-Neto J;Cerdán ME;González-Siso MI;Gombert AK
尽管马氏克鲁维酵母在生物过程中具有良好的生理特性，但与其近亲乳酸克鲁维酵母相比，它作为异源蛋白生产宿主的应用一直非常有限。本研究利用三种不同的表达系统，将黑曲霉葡萄糖氧化酶(GOx)的模式蛋白克隆到马氏克氏菌CBS 6556和乳酸克雷氏菌CBS 2359中。我们的目的是验证每个表达系统将如何影响蛋白质的表达、分泌/定位、翻译后修饰和生化特性。利用INU1启动子和终止子驱动外源基因的表达，以及INU1前前体序列驱动酶的分泌，获得了最高的Gox表达水平(每克干细胞重1552个单位的分泌蛋白)。在所有情况下，Gox主要分泌，留在周质空间或培养上清液中。使用来自酿酒酵母的遗传元件来驱动异源蛋白的表达导致乳酸克雷氏菌的表达水平高于马尔克森氏酵母，而使用INU1遗传元件则明显地导致相反的结果。Gox的生化特性证实了该蛋白的正确表达，并表明马氏克雷伯氏菌具有高糖化该蛋白的趋势，与其他酵母的情况类似，尽管这种趋势似乎比乳酸克雷伯氏菌和酿酒酵母要小。Gox的高糖基化似乎不影响其与底物的亲和力，也不影响其活性。综上所述，我们的结果表明，马氏克氏杆菌确实是表达异源蛋白的良好宿主，不仅因为它的生理特性，而且因为它正确地分泌和折叠这些蛋白。
In spite of its advantageous physiological properties for bioprocess applications, the use of the yeast Kluyveromyces marxianus as a host for heterologous protein production has been very limited, in constrast to its close relative Kluyveromyces lactis. In the present work, the model protein glucose oxidase (GOX) from Aspergillus niger was cloned into K. marxianus CBS 6556 and into K. lactis CBS 2359 using three different expression systems. We aimed at verifying how each expression system would affect protein expression, secretion/localization, post-translational modification, and biochemical properties. The highest GOX expression levels (1552 units of secreted protein per gram dry cell weight) were achieved using an episomal system, in which the INU1 promoter and terminator were used to drive heterologous gene expression, together with the INU1 prepro sequence, which was employed to drive secretion of the enzyme. In all cases, GOX was mainly secreted, remaining either in the periplasmic space or in the culture supernatant. Whereas the use of genetic elements from Saccharomyces cerevisiae to drive heterologous protein expression led to higher expression levels in K. lactis than in K. marxianus, the use of INU1 genetic elements clearly led to the opposite result. The biochemical characterization of GOX confirmed the correct expression of the protein and showed that K. marxianus has a tendency to hyperglycosylate the protein, in a similar way as already observed for other yeasts, although this tendency seems to be smaller than the one of e.g. K. lactis and S. cerevisiae. Hyperglycosylation of GOX does not seem to affect its affinity for the substrate, nor its activity. Taken together, our results indicate that K. marxianus is indeed a good host for the expression of heterologous proteins, not only for its physiological properties, but also because it correctly secretes and folds these proteins.
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影响因子：
3.2
作者：
Madzak, C;Otterbein, L;Beckerich, JM
通讯作者：
Beckerich, JM
影响因子：
3.2
作者：
ITO, H;FUKUDA, Y;KIMURA, A
通讯作者：
KIMURA, A
影响因子：
3.2
作者：
Bellaver, LH;de Carvalho, NMB;Gombert, AK
通讯作者：
Gombert, AK
影响因子：
5.4
作者：
Groeneveld, Philip;Stouthamer, Adriaan H.;Westerhoff, Hans V.
通讯作者：
Westerhoff, Hans V.
影响因子：
2.5
作者：
BERGKAMP, RJM;KOOL, IM;PLANTA, RJ
通讯作者：
PLANTA, RJ