caa a22 4500 465758088 CHVBK 20180323111852.0 cr unu---uuuuu 170327e19900801xx s 000 0 eng 10.1007/BF00312596 doi (NATIONALLICENCE)springer-10.1007/BF00312596 The influence of GAP promoter variants on hirudin production, average plasmid copy number and cell growth in Saccharomyces cerevisiae [Elektronische Daten] [M. Janes, B. Meyhack, W. Zimmermann, A. Hinnen] Summary: 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 μ 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. Springer-Verlag, 1990 Heterologous gene expression nationallicence Plasmid copy number nationallicence GAP promoter nationallicence Hirudin nationallicence Janes M. Biotechnology Department, Ciba-Geigy AG, K-681.1.44, P.O. Box, CH-4002, Basel, Switzerland aut Meyhack B. Biotechnology Department, Ciba-Geigy AG, K-681.1.44, P.O. Box, CH-4002, Basel, Switzerland aut Zimmermann W. Biotechnology Department, Ciba-Geigy AG, K-681.1.44, P.O. Box, CH-4002, Basel, Switzerland aut Hinnen A. Biotechnology Department, Ciba-Geigy AG, K-681.1.44, P.O. Box, CH-4002, Basel, Switzerland aut Current Genetics Springer-Verlag 18/2(1990-08-01), 97-103 0172-8083 18:2<97 1990 18 294 https://doi.org/10.1007/BF00312596 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/BF00312596 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Janes M. Biotechnology Department, Ciba-Geigy AG, K-681.1.44, P.O. Box, CH-4002, Basel, Switzerland aut NATIONALLICENCE 700 1- Meyhack B. Biotechnology Department, Ciba-Geigy AG, K-681.1.44, P.O. Box, CH-4002, Basel, Switzerland aut NATIONALLICENCE 700 1- Zimmermann W. Biotechnology Department, Ciba-Geigy AG, K-681.1.44, P.O. Box, CH-4002, Basel, Switzerland aut NATIONALLICENCE 700 1- Hinnen A. Biotechnology Department, Ciba-Geigy AG, K-681.1.44, P.O. Box, CH-4002, Basel, Switzerland aut NATIONALLICENCE 773 0- Current Genetics Springer-Verlag 18/2(1990-08-01), 97-103 0172-8083 18:2<97 1990 18 294 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer