Biomedical subjects

Michael Sauer

Publications and source records attributed to Michael Sauer.

17 recordsLinked to original sources

PubMed2006

Canalization of auxin flow by Aux/IAA-ARF-dependent feedback regulation of PIN polarity.

Plant development is characterized by a profound ability to regenerate and form tissues with new axes of polarity. An unsolved question concerns how the position within a tissue and cues from neighboring cells are integrated to specify the polarity of individual cells. The canalization hypothesis proposes a feedback effect of the phytohormone auxin on the directionality of intercellular auxin flow as a means to polarize tissues. Here we identify a cellular and molecular mechanism for canalization. Local auxin application, wounding, or auxin accumulation during de novo organ formation lead to rearrangements in the subcellular polar localization of PIN auxin transport components. This auxin effect on PIN polarity is cell-specific, does not depend on PIN transcription, and involves the Aux/IAA-ARF (indole-3-acetic acid-auxin response factor) signaling pathway. Our data suggest that auxin acts as polarizing cue, which links individual cell polarity with tissue and organ polarity through control of PIN polar targeting. This feedback regulation provides a conceptual framework for polarization during multiple regenerative and patterning processes in plants.

Arabidopsis↗

PubMed2006

High-gain Brillouin amplification: an analytical approach.

We derive a closed-form approximate analytical formula for the gain of a Brillouin fiber amplifier operating in the depleted pump regime. We take into account the material loss of the medium, which is a strong effect in optical fibers. We validate obtained results numerically and experimentally. The presented analysis can be used to accurately predict the performance of high-gain Brillouin fiber amplifiers.

Journal Article↗

PubMed2006

Lactate production yield from engineered yeasts is dependent from the host background, the lactate dehydrogenase source and the lactate export.

BACKGROUND: Metabolic pathway manipulation for improving the properties and the productivity of microorganisms is becoming a well established concept. For the production of important metabolites, but also for a better understanding of the fundamentals of cell biology, detailed studies are required. In this work we analysed the lactate production from metabolic engineered Saccharomyces cerevisiae cells expressing a heterologous lactate dehydrogenase (LDH) gene. The LDH gene expression in a budding yeast cell introduces a novel and alternative pathway for the NAD+ regeneration, allowing a direct reduction of the intracellular pyruvate to lactate, leading to a simultaneous accumulation of lactate and ethanol. RESULTS: Four different S. cerevisiae strains were transformed with six different wild type and one mutagenised LDH genes, in combination or not with the over-expression of a lactate transporter. The resulting yield values (grams of lactate produced per grams of glucose consumed) varied from as low as 0,0008 to as high as 0.52 g g-1. In this respect, and to the best of our knowledge, higher redirections of the glycolysis flux have never been obtained before without any disruption and/or limitation of the competing biochemical pathways. CONCLUSION: In the present work it is shown that the redirection of the pathway towards the lactate production can be strongly modulated by the genetic background of the host cell, by the source of the heterologous Ldh enzyme, by improving its biochemical properties as well as by modulating the export of lactate in the culture media.

Journal Article↗

PubMed2006

A molecular framework for plant regeneration.

Plants and some animals have a profound capacity to regenerate organs from adult tissues. Molecular mechanisms for regeneration have, however, been largely unexplored. Here we investigate a local regeneration response in Arabidopsis roots. Laser-induced wounding disrupts the flow of auxin-a cell-fate-instructive plant hormone-in root tips, and we demonstrate that resulting cell-fate changes require the PLETHORA, SHORTROOT, and SCARECROW transcription factors. These transcription factors regulate the expression and polar position of PIN auxin efflux-facilitating membrane proteins to reconstitute auxin transport in renewed root tips. Thus, a regeneration mechanism using embryonic root stem-cell patterning factors first responds to and subsequently stabilizes a new hormone distribution.

Arabidopsis↗

PubMed2006

Cloning, disruption and protein secretory phenotype of the GAS1 homologue of Pichia pastoris.

The aim of the study was the identification, cloning and disruption of the GAS1 homologue of Pichia pastoris. Gas1p is a glycoprotein anchored to the outer layer of the plasma membrane through a glycosylphosphatidylinositol (GPI) anchor. Gas1p is a beta-1,3-glucanosyltransglycosylase (EC 2.4.1.-). This cross-linking enzyme highly affects the structure and permeability of the yeast cell wall. The gene coding for the GAS1 homologue of P. pastoris was cloned by PCR, and its functionality was proven in a Saccharomyces cerevisiae GAS1 null mutant. Based on the nucleotide sequence information of the P. pastoris GAS1 homologue, a disruption cassette was constructed for the knockout of the GAS1 in P. pastoris. The morphology of DeltaGAS1 P. pastoris was identical to that of S. cerevisiae GAS1 mutants. Finally, the impact of GAS1 disruption on secretion of three recombinant model proteins in P. pastoris, human trypsinogen, human serum albumin and Rhizopus oryzae lipase, was evaluated. While the disruption had no effect on the secretion of trypsinogen and albumin, the amount of lipase released from the cells was doubled.

Amino Acid Sequence↗

PubMed2006

Improvement of lactic acid production in Saccharomyces cerevisiae by cell sorting for high intracellular pH.

Yeast strains expressing heterologous L-lactate dehydrogenases can produce lactic acid. Although these microorganisms are tolerant of acidic environments, it is known that at low pH, lactic acid exerts a high level of stress on the cells. In the present study we analyzed intracellular pH (pHi) and viability by staining with cSNARF-4F and ethidium bromide, respectively, of two lactic-acid-producing strains of Saccharomyces cerevisiae, CEN.PK m850 and CEN.PK RWB876. The results showed that the strain producing more lactic acid, CEN.PK m850, has a higher pHi. During batch culture, we observed in both strains a reduction of the mean pHi and the appearance of a subpopulation of cells with low pHi. Simultaneous analysis of pHi and viability proved that the cells with low pHi were dead. Based on the observation that the better lactic-acid-producing strain had a higher pHi and that the cells with low pHi were dead, we hypothesized that we might find better lactic acid producers by screening for cells within the highest pHi range. The screening was performed on UV-mutagenized populations through three consecutive rounds of cell sorting in which only the viable cells within the highest pHi range were selected. The results showed that lactic acid production was significantly improved in the majority of the mutants obtained compared to the parental strains. The best lactic-acid-producing strain was identified within the screening of CEN.PK m850 mutants.

Culture Media↗

PubMed2005

Maintenance of embryonic auxin distribution for apical-basal patterning by PIN-FORMED-dependent auxin transport in Arabidopsis.

Molecular mechanisms of pattern formation in the plant embryo are not well understood. Recent molecular and cellular studies, in conjunction with earlier microsurgical, physiological, and genetic work, are now starting to define the outlines of a model where gradients of the signaling molecule auxin play a central role in embryo patterning. It is relatively clear how these gradients are established and interpreted, but how they are maintained is still unresolved. Here, we have studied the contributions of auxin biosynthesis, conjugation, and transport pathways to the maintenance of embryonic auxin gradients. Auxin homeostasis in the embryo was manipulated by region-specific conditional expression of indoleacetic acid-tryptophan monooxygenase or indoleacetic acid-lysine synthetase, bacterial enzymes for auxin biosynthesis or conjugation. Neither manipulation of auxin biosynthesis nor of auxin conjugation interfered with auxin gradients and patterning in the embryo. This result suggests a compensatory mechanism for buffering auxin gradients in the embryo. Chemical and genetic inhibition revealed that auxin transport activity, in particular that of the PIN-FORMED1 (PIN1) and PIN4 proteins, is a major factor in the maintenance of these gradients.

Arabidopsis↗

PubMed2005

Reduced hydroperoxidase (HPI and HPII) activity in the Deltafur mutant contributes to increased sensitivity to UVA radiation in Escherichia coli.

In Escherichia coli, Deltafur (ferric uptake regulator) mutants are hypersensitive to various oxidative agents, including UVA radiation (400-315 nm). Studies suggest that UVA radiation mediates its biological effects on bacteria via oxidative mechanisms that lead to reactive oxygen species, including the superoxide anion radical (O2.-), hydroxyl radical (HO.), hydrogen peroxide (H2O2) and singlet oxygen (1O2). There is accumulating evidence that Fur may play an important role in the defense against UVA radiation. In addition to regulating almost all genes directly involved in iron acquisition, Fur also regulates the expression of manganese and iron superoxide dismutase (MnSOD, FeSOD), key enzymes in the defense against oxygen toxicity in E. coli. In Deltafur mutants, there is a complete absence of FeSOD. Previous results suggest that the native iron chelating agent, enterobactin, which exists in increased levels in Deltafur mutants, is an endogenous chromophore for UVA, releasing Fe2+ into the cytoplasm to catalyze the production of highly reactive hydroxyl radicals. We now report that the hypersensitivity of Deltafur mutants to UVA irradiation is associated with reduced hydroperoxidase I (HPI) and hydroperoxidase II (HPII) activity, and is associated with a decrease in the transcription of katE and katG genes. The observed decrease in HPII activity in Deltafur mutants is also associated with reduced rpoS gene transcription. This study provides additional evidence that the Fur gene product, in addition to its known regulatory effect on the expression of SOD and iron uptake mechanisms, also regulates HPI and HPII activity levels in E. coli. An H2O2-inducible antioxidant defense system leading to an increase in HPI activity, is unaltered in Deltafur mutants.

Bacterial Proteins↗

PubMed2005

Intracellular pH distribution in Saccharomyces cerevisiae cell populations, analyzed by flow cytometry.

Intracellular pH has an important role in the maintenance of the normal functions of yeast cells. The ability of the cell to maintain this pH homeostasis also in response to environmental changes has gained more and more interest in both basic and applied research. In this study we describe a protocol which allows the rapid determination of the intracellular pH of Saccharomyces cerevisiae cells. The method is based on flow cytometry and employs the pH-dependent fluorescent probe carboxy SNARF-4F. The protocol attempts to minimize the perturbation of the system under study, thus leading to accurate information about the physiological state of the single cell. Moreover, statistical analysis performed on major factors that may influence the final determination supported the validity of the optimized protocol. The protocol was used to investigate the effect of external pH on S. cerevisiae cells incubated in buffer. The results obtained showed that stationary cells are better able than exponentially grown cells to maintain their intracellular pH homeostasis independently of external pH changes. Furthermore, analysis of the intracellular pH distribution within the cell populations highlighted the presence of subpopulations characterized by different intracellular pH values. Notably, a different behavior was observed for exponentially grown and stationary cells in terms of the appearance and development of these subpopulations as a response to a changing external pH.

Benzopyrans↗

PubMed2005

Recombinant protein production in yeasts.

Recombinant DNA (rDNA) technologies (genetic, protein, and metabolic engineering) allow the production of a wide range of peptides, proteins, and biochemicals from naturally nonproducing cells. These technologies, now approx 25 yr old, have become one of the most important technologies developed in the twentieth century. Pharmaceutical products and industrial enzymes were the first biotech products on the world market made by means of rDNA. Despite important advances in rDNA applications in mammalian cells, yeasts still represent attractive hosts for the production of heterologous proteins. In this review we summarize advantages and limitations of the main and most promising yeast hosts.

Fermentation↗

PubMed2004

Differential gene expression in recombinant Pichia pastoris analysed by heterologous DNA microarray hybridisation.

BACKGROUND: Pichia pastoris is a well established yeast host for heterologous protein expression, however, the physiological and genetic information about this yeast remains scanty. The lack of a published genome sequence renders DNA arrays unavailable, thereby hampering more global investigations of P. pastoris from the beginning. Here, we examine the suitability of Saccharomyces cerevisiae DNA microarrays for heterologous hybridisation with P. pastoris cDNA. RESULTS: We could show that it is possible to obtain new and valuable information about transcriptomic regulation in P. pastoris by probing S. cerevisiae DNA microarrays. The number of positive signals was about 66 % as compared to homologous S. cerevisiae hybridisation, and both the signal intensities and gene regulations correlated with high significance between data obtained from P. pastoris and S. cerevisiae samples. The differential gene expression patterns upon shift from glycerol to methanol as carbon source were investigated in more detail. Downregulation of TCA cycle genes and a decrease of genes related to ribonucleotide and ribosome synthesis were among the major effects identified. CONCLUSIONS: We could successfully demonstrate that heterologous microarray hybridisations allow deep insights into the transcriptomic regulation processes of P. pastoris. The observed downregulation of TCA cycle and ribosomal synthesis genes correlates to a significantly lower specific growth rate during the methanol feed phase.

Journal Article↗

PubMed2004

Stress in recombinant protein producing yeasts.

It is well established today that heterologous overexpression of proteins is connected with different stress reactions. The expression of a foreign protein at a high level may either directly limit other cellular processes by competing for their substrates, or indirectly interfere with metabolism, if their manufacture is blocked, thus inducing a stress reaction of the cell. Especially the unfolded protein response (UPR) in Saccharomyces cerevisiae (as well as some other yeasts) is well documented, and its role for the limitation of expression levels is discussed. One potential consequence of endoplasmatic reticulum folding limitations is the ER associated protein degradation (ERAD) involving retrotranslocation and decay in the cytosol. High cell density fermentation, the typical process design for recombinant yeasts, exerts growth conditions that deviate far from the natural environment of the cells. Thus, different environmental stresses may be exerted on the host. High osmolarity, low pH and low temperature are typical stress factors. Whereas the molecular pathways of stress responses are well characterized, there is a lack of knowledge concerning the impact of stress responses on industrial production processes. Accordingly, most metabolic engineering approaches conducted so far target at the improvement of protein folding and secretion, whereas only few examples of cell engineering against general stress sensitivity were published. Apart from discussing well-documented stress reactions of yeasts in the context of heterologous protein production, some more speculative topics like quorum sensing and apoptosis are addressed.

Biotechnology↗

PubMed2004

The yeast Zygosaccharomyces bailii: a new host for heterologous protein production, secretion and for metabolic engineering applications.

Molecular tools for the production of heterologous proteins and metabolic engineering applications of the non-conventional yeast Zygosaccharomyces bailii were developed. The combination of Z. bailii's resistance to relatively high temperature, osmotic pressure and low pH values, with a high specific growth rate renders this yeast potentially interesting for exploitation for biotechnological purposes as well as for the understanding of the biological phenomena and mechanisms underlying the respective resistances. Looking forward to these potential applications, here we present the tools required for the production and the secretion of different heterologous proteins, and one example of a metabolic engineering application of this non-conventional yeast, employing the newly developed molecular tools.

Cloning, Molecular↗

PubMed2004

In vitro culture of Arabidopsis embryos within their ovules.

Embryogenesis of flowering plants establishes a basic body plan with apical-basal, radial and bilateral patterns from the single-celled zygote. Arabidopsis embryogenesis exhibits a nearly invariant cell division pattern and therefore is an ideal system for studies of early plant development. However, plant embryos are difficult to access for experimental manipulation, as they develop deeply inside maternal tissues. Here we present a method for the culture of zygotic Arabidopsis embryos in vitro. The technique omits excision of the embryo by culturing the entire ovule, thus greatly facilitating the time and effort involved. It enables external manipulation of embryo development and culture from the earliest developmental stages up to maturity. Administration of various chemical treatments as well as the use of different molecular markers is demonstrated together with standard techniques for visualizing gene expression and protein localization in in vitro cultivated embryos. The presented set of techniques allows for so far unavailable molecular physiology approaches in the study of early plant development.

Arabidopsis↗

PubMed2004

Production of L-ascorbic acid by metabolically engineered Saccharomyces cerevisiae and Zygosaccharomyces bailii.

Yeasts do not possess an endogenous biochemical pathway for the synthesis of vitamin C. However, incubated with l-galactose, L-galactono-1,4-lactone, or L-gulono-1,4-lactone intermediates from the plant or animal pathway leading to l-ascorbic acid, Saccharomyces cerevisiae and Zygosaccharomyces bailii cells accumulate the vitamin intracellularly. Overexpression of the S. cerevisiae enzymes d-arabinose dehydrogenase and D-arabinono-1,4-lactone oxidase enhances this ability significantly. In fact, the respective recombinant yeast strains even gain the capability to accumulate the vitamin in the culture medium. An even better result is obtainable by expression of the plant enzyme L-galactose dehydrogenase from Arabidopsis thaliana. Budding yeast cells overexpressing the endogenous D-arabinono-1,4-lactone oxidase as well as L-galactose dehydrogenase are capable of producing about 100 mg of L-ascorbic acid liter(-1), converting 40% (wt/vol) of the starting compound L-galactose.

Arabidopsis↗

PubMed2003

Local, efflux-dependent auxin gradients as a common module for plant organ formation.

Plants, compared to animals, exhibit an amazing adaptability and plasticity in their development. This is largely dependent on the ability of plants to form new organs, such as lateral roots, leaves, and flowers during postembryonic development. Organ primordia develop from founder cell populations into organs by coordinated cell division and differentiation. Here, we show that organ formation in Arabidopsis involves dynamic gradients of the signaling molecule auxin with maxima at the primordia tips. These gradients are mediated by cellular efflux requiring asymmetrically localized PIN proteins, which represent a functionally redundant network for auxin distribution in both aerial and underground organs. PIN1 polar localization undergoes a dynamic rearrangement, which correlates with establishment of auxin gradients and primordium development. Our results suggest that PIN-dependent, local auxin gradients represent a common module for formation of all plant organs, regardless of their mature morphology or developmental origin.

Arabidopsis↗

PubMed2003

Efflux-dependent auxin gradients establish the apical-basal axis of Arabidopsis.

Axis formation occurs in plants, as in animals, during early embryogenesis. However, the underlying mechanism is not known. Here we show that the first manifestation of the apical-basal axis in plants, the asymmetric division of the zygote, produces a basal cell that transports and an apical cell that responds to the signalling molecule auxin. This apical-basal auxin activity gradient triggers the specification of apical embryo structures and is actively maintained by a novel component of auxin efflux, PIN7, which is located apically in the basal cell. Later, the developmentally regulated reversal of PIN7 and onset of PIN1 polar localization reorganize the auxin gradient for specification of the basal root pole. An analysis of pin quadruple mutants identifies PIN-dependent transport as an essential part of the mechanism for embryo axis formation. Our results indicate how the establishment of cell polarity, polar auxin efflux and local auxin response result in apical-basal axis formation of the embryo, and thus determine the axiality of the adult plant.

Arabidopsis↗