Janes M, Meyhack B, Zimmermann W, Hinnen A
Ciba-Geigy AG, Biotechnology Department, Basel, Switzerland.
Curr Genet. 1990 Aug;18(2):97-103. doi: 10.1007/BF00312596.
The yeast Saccharomyces cerevisiae has been engineered to synthesize and secrete desulfato-hirudin (hirudin), a thrombin inhibitor from the leech Hirudo medicinalis. The synthetic gene coding for hirudin was expressed constitutively under the control of four size-variants of the yeast glyceraldehyde-3-phosphate dehydrogenase promoter (GAP) and cloned into a 2 mu based multicopy yeast vector. The constitutive action of the four promoter variants was confirmed by demonstrating that the expression and secretion of hirudin is growth-related. The different efficiencies of the promoter variants not only affected hirudin expression but also led to changes in several cellular parameters, such as cell growth, average plasmid copy number and plasmid stability. The observed changes show that yeast cells establish a specific equilibrium for each promoter variant. We conclude, that the adjustment of cellular parameters in response to the expression levels of a heterologous protein is regulated by two counteracting selective forces: (1) the need for complementation of the auxotrophic host marker by the plasmid-encoded selection gene which, in the case of dLEU2, requires several plasmid copies; and (2) a selective advantage of cells with a lower copy number enabling them to escape the burden of heterologous protein production.
酿酒酵母已被改造用于合成和分泌去硫酸化水蛭素（水蛭素），这是一种来自医用水蛭的凝血酶抑制剂。编码水蛭素的合成基因在酵母甘油醛-3-磷酸脱氢酶启动子（GAP）的四种大小变体的控制下组成型表达，并被克隆到基于2μm的多拷贝酵母载体中。通过证明水蛭素的表达和分泌与生长相关，证实了四种启动子变体的组成型作用。启动子变体的不同效率不仅影响水蛭素的表达，还导致了几个细胞参数的变化，如细胞生长、平均质粒拷贝数和质粒稳定性。观察到的变化表明，酵母细胞为每个启动子变体建立了特定的平衡。我们得出结论，细胞参数响应异源蛋白表达水平的调节受到两种相互抵消的选择力的影响：（1）质粒编码的选择基因对营养缺陷型宿主标记进行互补的需要，在dLEU2的情况下，这需要几个质粒拷贝；（2）较低拷贝数的细胞具有选择性优势，使其能够摆脱异源蛋白产生的负担。
