Tilman Achstetter

Probing the limits of expression levels by varying promoter strength and plasmid copy number in Saccharomyces cerevisiae

Heterologous gene expression levels were measured in yeast using the Escherichia coli gusA gene (encoding beta-D-glucuronidase) as a reporter. The influence of two major parameters, promoter activity and plasmid copy number, was studied. (1) Promoters used in this study ranged from the very weak constitutive KEX2, the regulated CYC1 and PGK and the mating type-specific MF alpha 1 to the strong constitutive TEF1 and TDH promoters. Using centromeric vectors, gusA expression levels varied within three orders of magnitude. (2) Plasmid copy number was changed by shifting from a monocopy (centromeric plasmid) over a moderate copy number (2 mu-based plasmid) to a high copy number (2 mu associated with the URA3-d selection marker). gusA expression levels increased relatively with plasmid copy number in all cases studied, but did not exceed the equivalent of 2% of total soluble yeast proteins. Coupling these variables, a 5-log range in gene expression levels was covered. Taken together, these results provide a framework which allows a comparison of existing and new promoters. This framework will be useful for expressing genes to required levels.

Characterization of Recombinant Adrenodoxin Reductase Homologue (Arh1p) from Yeast IMPLICATION IN IN VITRO CYTOCHROME P45011β MONOOXYGENASE SYSTEM

The mammalian electron transfer chain of mitochondrial cytochrome P450 forms involved in steroidogenesis includes very specific proteins, namely adrenodoxin reductase and adrenodoxin. Adrenodoxin reductase transfers electrons from NADPH to adrenodoxin, which subsequently donates them to the cytochrome P450 forms. The Saccharomyces cerevisiae ARH1 gene product (Arh1p) presents homology to mammalian adrenodoxin reductase. We demonstrate the capacity of recombinant Arh1p, made inEscherichia coli, to substitute for its mammalian homologue in ferricyanide, cytochrome c reduction, and, more importantly, in vitro 11β-hydroxylase assays. Electrons could be transferred from NADPH and NADH as measured in the cytochrome c reduction assay. ApparentK m values were determined to be 0.5, 0.6, and 0.1 μm for NADPH, NADH, and bovine adrenodoxin, respectively. These values differ slightly from those of mammalian adrenodoxin reductase, except for NADH, which is a very poor electron donor to the mammalian protein. Subcellular fractionation studies have localized Arh1p to the inner membrane of yeast mitochondria. The biological function of Arh1p remains unknown, and to date, no mitochondrial cytochrome P450 has been identified. ARH1 is, however, essential for yeast viability because an ARH1 gene disruption is lethal not only in aerobic growth conditions but also, surprisingly enough, during fermentation.

New proteolytic enzymes in yeast.

Abstract Yeast mutants lacking three proteolytic enzymes—proteinase B, carboxypeptidase Y, and carboxypeptidase S—have been constructed. Search for new proteolytic activities in these mutants with the aid of chromogenic peptide substrates developed for serum proteinases led to the detection of new proteolytic activities, active in the neutral pH range. Sephadex chromatography of a 100,000g supernate of mutant extracts, tests against four different substrates, and partial characterization of their sensitivity to various inhibitors indicate multiple activities. Two activities, called proteinase M and proteinase P, were found in the sedimentable membranous fraction of mutant extracts.

11β-Hydroxylase Activity in Recombinant Yeast Mitochondria

In mammals, the final 11 beta-hydroxylation step of the hydrocortisone biosynthesis pathway is performed by a mitochondrial enzyme, namely cytochrome P-450(11 beta), together with the electron carriers adrenodoxin and NADPH adrenodoxin oxidoreductase. Successful production of a functional steroid 11 beta-hydroxylase activity was obtained in recombinant yeast in vivo. This conversion was achieved by coexpression of a mitochondrially targeted adrenodoxin and a modified bovine P-450(11 beta) whose natural presequence was replaced by a yeast presequence, together with an unexpected yeast endogenous NADPH-adrenodoxin-reductase-like activity. Adrenodoxin and P-450(11 beta) behave as a mitochondrial matrix and membrane protein, respectively. Saccharomyces cerevisiae apparently produces a mitochondrial protein which is capable of transferring electrons to bovine adrenodoxin, which in turn transfers the electrons to P-450(11 beta). The endogenous adrenodoxin oxidoreductase gains electrons specifically from NADPH. The notion that a yeast microsomal NADPH P-450 oxidoreductase can transfer electrons to mammalian microsomal P-450s can be extended to mitochondria, where an NADPH adrenodoxin oxidoreductase protein transfers electrons to adrenodoxin and renders a mitochondrial mammalian P-450 functional in vivo. The physiological function of this yeast NADPH adrenodoxin oxidoreductase activity is not known.

Aminopeptidase Co, a new yeast peptidase

Abstract A new aminopeptidase — aminopeptidase Co — has been detected in the yeast Saccharomyces cerevisiae . The enzyme is only active in the presence of Co2+ions. Zn2+- and Mn2+ions are inhibitory. The enzyme activity is also inhibited by chelating agents. Of the p-nitroanilide derivatives tested only those containing basic amino acids are cleaved.

Proteolysis in eucaryotic cells: Aminopeptidases and dipeptidyl aminopeptidases of yeast revisited

Using nine different L-aminoacyl-4-nitroanilides and four different dipeptidyl-4-nitroanilides, aminopeptidases and dipeptidyl aminopeptidases active at pH 7.5 and (or) pH 5.5 in logarithmically growing and stationary-phase cells of Saccharomyces cerevisiae were searched for. Ion-exchange chromatography was used to separate the proteins of the soluble cell extract. Besides the three already-characterized aminopeptidases--aminopeptidase I (P. Matile, A. Wiemken, and W. Guyer (1971) Planta (Berlin) 96, 43-53; J. Frey and K. H. Röhm (1978) Biochim. Biophys. Acta 527, 31-41), aminopeptidase II (J. Frey and K. H. Röhm (1978) Biochim. Biophys. Acta 527, 31-41; J. Knüver (1982) Thesis, Fachbereich Chemie, Marburg, FRG), and aminopeptidase Co (T. Achstetter, C. Ehmann, and D. H. Wolf (1982) Biochem. Biophys. Res. Commun. 109, 341-347)--12 additional aminopeptidase activities are found in soluble cell extracts eluting from the ion-exchange column. These activities differ from the characterized aminopeptidases in one or more of the parameters such as charge, size, substrate specificity, inhibition pattern, pH optimum for activity and regulation. Also, a particulate aminopeptidase, called aminopeptidase P, is found in the nonsoluble fraction of disintegrated cells. Besides the described particulate X-prolyl-dipeptidyl aminopeptidase (M. P. Suarez Rendueles, J. Schwencke, N. Garcia-Alvarez and S. Gascon (1981) FEBS Lett. 131, 296-300), three additional dipeptidyl aminopeptidase activities of different substrate specificities are found in the soluble extract.

Yeast pheromone α-factor is synthesized as a high molecular weight precursor

Summary The sex pheromone α-factor of Saccharomyces cerevisiae , a tridecapeptide of approx. 1,700 molecular weight, was found to be synthesized in vivo as a high molecular weight precursor of M r = 28,000. Inhibition of N-linked glycosylation by tunicamycin leads to three precursor species of lower molecular weight indicating three carbohydrate residues linked to the α-factor precursor molecule. A molecular weight of 18,000 was determined for the unglycosylated molecule.

A set of isomeric episomal plasmids for systematic examination of mitotic stability in Saccharomyces cerevisiae

Yeast episomal shuttle vectors (YEp type) are commonly used in fundamental research and biotechnology whenever elevated product levels are desired. Their instability, however, poses an impediment not only in industrial scale fermentation. In order to analyse instability which might be linked to plasmid structure, a series of YEp type plasmids that are identical in size has been assembled, differing only in the overall arrangement of the fragments used. The performance of the eight plasmid isoforms was studied with respect to mitotic stability. While transformation efficiency in two laboratory strains of Saccharomyces cerevisiae does not differ dramatically between the eight plasmids, the plasmids do not, however, perform equally well in terms of segregational stability. Although stable at about 90% plasmid‐bearing cells in selective medium, under non‐selective conditions, three plasmid forms performed better than the other five with an up to 5.7‐fold higher stability as compared with the least favourable isoform. In a subset of four plasmids (including stable and unstable isoforms) copy numbers were determined. Furthermore the functionality of the selection marker was characterized with respect to plasmid‐derived relative HIS3 transcript levels. No significant differences in HIS3 transcript levels could be observed between strains carrying any one of the four plasmids. Ruling out copy number and performance of HIS3, the results indicate nevertheless that plasmid architecture has an impact on mitotic segregation in yeast and that construction of an expression vector should take into account that the plasmid backbone itself might already show a more or less favourable arrangement of its segments.