Research papers
An interlaboratory comparison of physiological and genetic properties of four Saccharomyces cerevisiae strains

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Abstract

To select a Saccharomyces cerevisiae reference strain amenable to experimental techniques used in (molecular) genetic, physiological and biochemical engineering research, a variety of properties were studied in four diploid, prototrophic laboratory strains. The following parameters were investigated: 1) maximum specific growth rate in shake-flask cultures; 2) biomass yields on glucose during growth on defined media in batch cultures and steady-state chemostat cultures under controlled conditions with respect to pH and dissolved oxygen concentration; 3) the critical specific growth rate above which aerobic fermentation becomes apparent in glucose-limited accelerostat cultures; 4) sporulation and mating efficiency; and 5) transformation efficiency via the lithium-acetate, bicine, and electroporation methods. On the basis of physiological as well as genetic properties, strains from the CEN.PK family were selected as a platform for cell-factory research on the stoichiometry and kinetics of growth and product formation.

Introduction

Optimization of the yeast cell factory for industrial applications requires a multidisciplinary effort of (molecular) geneticists, physiologists, and biochemical engineers. Each of these disciplines imposes specific requirements on the yeast strains used for experimental work. Required properties, that often seem to be noncompatible in a single strain, include high transformation efficiency, fast growth in defined mineral media under controlled conditions, high rates of respiratory growth in sugar-limited chemostat cultures, etc. (Table 1).

The conflicts of interest that may arise in selecting a suitable strain platform for multidisciplinary research are exemplified by Saccharomyces cerevisiae CBS 8066. This prototrophic, diploid wild-type strain has been extensively characterized with respect to its physiology [1], [2], [3], [4], [5]. However, strain CBS 8066 is heterozygous for a number of auxotrophic markers and poorly transformable, which is a major complication in integrated genetic/physiological studies.

As a result of their different immediate research goals, geneticists, physiologists and biochemical engineers use a variety of S. cerevisiae strains. Even within each single discipline, a multitude of strains are studied because, for obvious reasons, a particular research group tends to adhere to the “house strain” with which previous research has been carried out. It is evident that a common reference strain, and isogenic mutants derived from it, is of great value for studies on quantitative aspects of growth and metabolism. Such studies require the use of prototrophic strains and defined mineral media. Cultivation of strains carrying auxotrophic markers requires the addition of amino acids, purine or pyrimidine bases to the medium, which complicates physiological studies [6] and in some cases obscures changes in physiological properties caused by genetic modification. For example, S. cerevisiae mutants lacking the E1α subunit of the mitochondrial pyruvate-dehydrogenase complex exhibit a partial leucine requirement [7] and mutants lacking glucose-6-phosphate dehydrogenase require methionine [8]. These interesting effects would remain unnoticed in leucine/methionine auxotrophs or in complex media.

In view of the lack of consensus on the choice of a common reference strain for genetic, physiological, and engineering studies, we decided to characterize a number of laboratory strains that are currently in use in our laboratories. Emphasis was given to standard transformation tests and cultivation under well-defined conditions. The results obtained show that strains from the CEN.PK family offer an acceptable compromise between the criteria set by different research disciplines.

Section snippets

Strains

Four diploid, prototrophic laboratory strains were used in this study:

  • 1.

    Saccharomyces cerevisiae CBS8066. This strain was obtained from the Centraal Bureau voor Schimmelcultures (CBS, Delft, The Netherlands) and included because it has been extensively characterized with respect to its physiological properties [1], [2], [3], [4], [5]. A drawback of this diploid strain for genetic studies is that it is heterozygous for the auxotrophic recessive alleles his4, leu1, and met2 (H.Y. Steensma and J.

Physiological tests

The maximum specific growth rate in vitamin-supplemented mineral media was determined in shake-flask cultures via optical-density measurements. To obtain an impression of the reproducibility of these assays, the specific growth rate on glucose was estimated in all our 10 laboratories. A SD of ca. 20% was obtained for data collected in the various laboratories. However, duplicate experiments performed within one laboratory generally differed by less than 5%. From the results of these shake-flask

Physiological and genetic characteristics

In comparison with many other yeasts, the range of carbon substrates that support growth of S. cerevisiae is rather narrow. Even so, not all relevant substrates have been tested in this study. For example, oleate [22] was not included, as growth on this substrate is difficult to quantify. Furthermore, from the many nitrogen sources supporting growth of S. cerevisiae strains [23], only ammonia was tested. Nevertheless, the data presented in Table 2 show that strain X2180 is inferior compared to

Requests for CEN.PK Strains

Requests for CEN.PK strains [33] should not be addressed to the authors of this paper, but to Dr. P. Kötter, Institute of Microbiology, J.W. Goethe Universität, Marie-Curie-Strasse 9, D-60439 Frankfurt, Germany (e-mail [email protected]).

Acknowledgements

This work is part of the project “From Gene to Product in Yeast: a Quantitative Approach,” which is subsidized by the European Community (EC Framework IV Cell Factory Program). We acknowledge the contributions to the experimental work of K. Bangma, D. Bolzani, M. Ehlde, P. de Jong-Gubbels, and M. Luttik and the background information on CEN.PK strains provided by Dr. P. Kötter.

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    Prof. Dr. Manfred Rizzi deceased on 8 May, 1998.

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