Görgens Johann F, Passoth Volkmar, van Zyl Willem H, Knoetze Johannes H, Hahn-Hägerdal Bärbel
Department of Applied Microbiology, Lund University, Lund, Sweden.
FEMS Yeast Res. 2005 Apr;5(6-7):677-83. doi: 10.1016/j.femsyr.2004.12.003.
Heterologous endo-beta-1,4-xylanase was produced by Pichia stipitis under control of the hypoxia-inducible PsADH2-promoter in a high-cell-density culture. After promoter induction by a shift to oxygen limitation, different aeration rates (oxygen transfer rates) were applied while maintaining oxygen-limitation. Initially, enzyme production was higher in oxygen-limited cultures with high rates of oxygen transfer, although the maximum xylanase activity was not significantly influenced. Amino acid supplementation increased the production of the heterologous endo-beta-1,4-xylanase significantly in highly aerated oxygen-limited cultures, until glucose was depleted. A slight second induction of the promoter was observed in all cultures after the glucose had been consumed. The second induction was most obvious in amino acid-supplemented cultures with higher oxygen transfer rates during oxygen limitation. When such oxygen-limited cultures were shifted back to fully aerobic conditions, a significant re-induction of heterologous endo-beta-1,4-xylanase production was observed. Re-induction was accompanied by ethanol consumption. A similar protein production pattern was observed when cultures were first grown on ethanol as sole carbon source and subsequently glucose and oxygen limitation were applied. Thus, we present the first expression system in yeast with a sequential double-inducible promoter.
在高细胞密度培养中，树干毕赤酵母（Pichia stipitis）在缺氧诱导型PsADH2启动子的控制下产生了异源内切-β-1,4-木聚糖酶。通过转变为氧限制来诱导启动子后，在维持氧限制的同时应用了不同的通气速率（氧传递速率）。最初，在氧传递速率高的氧限制培养物中酶产量较高，尽管木聚糖酶的最大活性没有受到显著影响。在高通气量的氧限制培养物中，直到葡萄糖耗尽，氨基酸补充显著增加了异源内切-β-1,4-木聚糖酶的产量。在葡萄糖消耗后，在所有培养物中都观察到启动子的轻微二次诱导。二次诱导在氧限制期间氧传递速率较高的氨基酸补充培养物中最为明显。当这种氧限制培养物转回完全好氧条件时，观察到异源内切-β-1,4-木聚糖酶产量的显著再诱导。再诱导伴随着乙醇的消耗。当培养物首先以乙醇作为唯一碳源生长，随后施加葡萄糖和氧限制时，观察到类似的蛋白质产生模式。因此，我们展示了酵母中第一个具有顺序双诱导启动子的表达系统。
