Transcriptome analysis of a respiratory Saccharomyces cerevisiae strain suggests the expression of its phenotype is glucose insensitive and predominantly controlled by Hap4, Cat8 and Mig1

BACKGROUND: We previously described the first respiratory Saccharomyces cerevisiae strain, KOY.TM6*P, by integrating the gene encoding a chimeric hexose transporter, Tm6*, into the genome of an hxt null yeast. Subsequently we transferred this respiratory phenotype in the presence of up to 50 g/L glucose to a yeast strain, V5 hxt1-7Delta, in which only HXT1-7 had been deleted. In this study, we compared the transcriptome of the resultant strain, V5.TM6*P, with that of its wild-type parent, V5, at different glucose concentrations. RESULTS: cDNA array analyses revealed that alterations in gene expression that occur when transitioning from a respiro-fermentative (V5) to a respiratory (V5.TM6*P) strain, are very similar to those in cells undergoing a diauxic shift. We also undertook an analysis of transcription factor binding sites in our dataset by examining previously-published biological data for Hap4 (in complex with Hap2, 3, 5), Cat8 and Mig1, and used this in combination with verified binding consensus sequences to identify genes likely to be regulated by one or more of these. Of the induced genes in our dataset, 77% had binding sites for the Hap complex, with 72% having at least two. In addition, 13% were found to have a binding site for Cat8 and 21% had a binding site for Mig1. Unexpectedly, both the up- and down-regulation of many of the genes in our dataset had a clear glucose dependence in the parent V5 strain that was not present in V5.TM6*P. This indicates that the relief of glucose repression is already operable at much higher glucose concentrations than is widely accepted and suggests that glucose sensing might occur inside the cell. CONCLUSION: Our dataset gives a remarkably complete view of the involvement of genes in the TCA cycle, glyoxylate cycle and respiratory chain in the expression of the phenotype of V5.TM6*P. Furthermore, 88% of the transcriptional response of the induced genes in our dataset can be related to the potential activities of just three proteins: Hap4, Cat8 and Mig1. Overall, our data support genetic remodelling in V5.TM6*P consistent with a respiratory metabolism which is insensitive to external glucose concentrations.

Eukaryotic expression:Developments for structural proteomics

The production of sufficient quantities of protein is an essential prelude to a structure determination, but for many viral and human proteins this cannot be achieved using prokaryotic expression systems. Groups in the Structural Proteomics In Europe (SPINE) consortium have developed and implemented high-throughput (HTP) methodologies for cloning, expression screening and protein production in eukaryotic systems. Studies focused on three systems: yeast (Pichia pastoris and Saccharomyces cerevisiae), baculovirus-infected insect cells and transient expression in mammalian cells. Suitable vectors for HTP cloning are described and results from their use in expression screening and protein-production pipelines are reported. Strategies for co-expression, selenomethionine labelling (in all three eukaryotic systems) and control of glycosylation (for secreted proteins in mammalian cells) are assessed. © International Union of Crystallography, 2006.

Improved yields of full-length functional human FGF1 can be achieved using the methylotrophic yeast Pichia pastoris

We have produced human fibroblast growth factor 1 (hFGF1) in the methylotrophic yeast Pichia pastoris in order to obtain the large amounts of active protein required for subsequent functional and structural characterization. Four constructs were made to examine both intracellular and secreted expression, with variations in the location of the His6 tag at either end of the peptide. hFGF1 could be produced from all four constructs in shake flasks, but production was optimized by growing only the highest-yielding of these strains, which produced hFGF1 intracellularly, under tightly controlled conditions in a 3 L fermentor. One hundred and eight milligrams of pure protein was achieved per liter culture (corresponding to 0.68 mg of protein per gram of wet cells), the function of which was verified using NIH 3T3 cell cultures. This is a 30-fold improvement over previously reported yields of full-length hFGF1. © 2006 Elsevier Inc. All rights reserved.

Design of improved membrane protein production experiments:quantitation of the host response

Eukaryotic membrane proteins cannot be produced in a reliable manner for structural analysis. Consequently, researchers still rely on trial-and-error approaches, which most often yield insufficient amounts. This means that membrane protein production is recognized by biologists as the primary bottleneck in contemporary structural genomics programs. Here, we describe a study to examine the reasons for successes and failures in recombinant membrane protein production in yeast, at the level of the host cell, by systematically quantifying cultures in high-performance bioreactors under tightlydefined growth regimes. Our data show that the most rapid growth conditions of those chosen are not the optimal production conditions. Furthermore, the growth phase at which the cells are harvested is critical: We show that it is crucial to grow cells under tightly-controlled conditions and to harvest them prior to glucose exhaustion, just before the diauxic shift. The differences in membrane protein yields that we observe under different culture conditions are not reflected in corresponding changes in mRNA levels of FPS1, but rather can be related to the differential expression of genes involved in membrane protein secretion and yeast cellular physiology. Copyright © 2005 The Protein Society.

Role of hexose transport in control of glycolytic flux in Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae predominantly ferments glucose to ethanol at high external glucose concentrations, irrespective of the presence of oxygen. In contrast, at low external glucose concentrations and in the presence of oxygen, as in a glucose-limited chemostat, no ethanol is produced. The importance of the external glucose concentration suggests a central role for the affinity and maximal transport rates of yeast's glucose transporters in the control of ethanol production. Here we present a series of strains producing functional chimeras between the hexose transporters Hxt1 and Hxt7, each of which lias distinct glucose transport characteristics. The strains display a range of decreasing glycolytic rates resulting in a proportional decrease in ethanol production. Using these strains, we show for the first time that at high glucose levels, the glucose uptake capacity of wild-type S. cerevisiae does not control glycolytic flux during exponential batch growth. In contrast, our chimeric Hxt transporters control the rate of glycolysis to a high degree. Strains whose glucose uptake is mediated by these chimeric transporters will undoubtedly provide a powerful tool with which to examine in detail the mechanism underlying the switch between fermentation and respiration in S. cerevisiae and will provide new tools for the control of industrial fermentations.

Switching the mode of metabolism in the yeast Saccharomyces cerevisiae

The biochemistry of most metabolic pathways is conserved from bacteria to humans, although the control mechanisms are adapted to the needs of each cell type. Oxygen depletion commonly controls the switch from respiration to fermentation. However, Saccharomyces cerevisiae also controls that switch in response to the external glucose level. We have generated an S. cerevisiae strain in which glucose uptake is dependent on a chimeric hexose transporter mediating reduced sugar uptake. This strain shows a fully respiratory metabolism also at high glucose levels as seen for aerobic organisms, and switches to fermentation only when oxygen is lacking. These observations illustrate that manipulating a single step can alter the mode of metabolism. The novel yeast strain is an excellent tool to study the mechanisms underlying glucose-induced signal transduction. © 2004 European Molecular Biology Organization.

Increasing cell biomass in Saccharomyces cerevisiae increases recombinant protein yield:the use of a respiratory strain as a microbial cell factory

BACKGROUND: Recombinant protein production is universally employed as a solution to obtain the milligram to gram quantities of a given protein required for applications as diverse as structural genomics and biopharmaceutical manufacture. Yeast is a well-established recombinant host cell for these purposes. In this study we wanted to investigate whether our respiratory Saccharomyces cerevisiae strain, TM6*, could be used to enhance the productivity of recombinant proteins over that obtained from corresponding wild type, respiro-fermentative strains when cultured under the same laboratory conditions. RESULTS: Here we demonstrate at least a doubling in productivity over wild-type strains for three recombinant membrane proteins and one recombinant soluble protein produced in TM6* cells. In all cases, this was attributed to the improved biomass properties of the strain. The yield profile across the growth curve was also more stable than in a wild-type strain, and was not further improved by lowering culture temperatures. This has the added benefit that improved yields can be attained rapidly at the yeast's optimal growth conditions. Importantly, improved productivity could not be reproduced in wild-type strains by culturing them under glucose fed-batch conditions: despite having achieved very similar biomass yields to those achieved by TM6* cultures, the total volumetric yields were not concomitantly increased. Furthermore, the productivity of TM6* was unaffected by growing cultures in the presence of ethanol. These findings support the unique properties of TM6* as a microbial cell factory. CONCLUSIONS: The accumulation of biomass in yeast cell factories is not necessarily correlated with a proportional increase in the functional yield of the recombinant protein being produced. The respiratory S. cerevisiae strain reported here is therefore a useful addition to the matrix of production hosts currently available as its improved biomass properties do lead to increased volumetric yields without the need to resort to complex control or cultivation schemes. This is anticipated to be of particular value in the production of challenging targets such as membrane proteins.

Optical fibre Bragg grating recorded in TOPAS cyclic olefin copolymer

A report is presented on the inscription of a fibre Bragg grating into a microstructured polymer optical fibre fabricated from TOPAS cyclic olefin copolymer. This material offers two important advantages over poly (methyl methacrylate), which up to now has formed the basis for polymer fibre Bragg gratings: TOPAS has a much lower water affinity and has useful properties for biosensing. The grating had a Bragg wavelength of 1569 nm and a temperature sensitivity of -36.5±0.3 pm/°C.

870nm Bragg grating in single mode TOPAS microstructured polymer optical fibre

We report the fabrication and characterization of a fiber Bragg grating (FBG) with 870 nm resonance wavelength in a single-mode TOPAS microstructured polymer optical fiber (mPOF). The grating has been UV-written with the phasemask technique using a 325 nm HeCd laser. The static tensile strain sensitivity has been measured as 0.64 pm/µstrain, and the temperature sensitivity was -60 pm/°C. This is the first 870nm FBG and the first demonstration of a negative temperature response for the TOPAS FBG, for which earlier results have indicated a positive temperature response. The relatively low material loss of the fiber at this wavelength compared to that at longer wavelengths will considerably enhance the potential utility of the TOPAS FBG.

Polymer PCF Bragg grating sensors based on poly(methyl methacrylate) and TOPAS cyclic olefin copolymer

Fibre Bragg grating (FBG) sensors have been fabricated in polymer photonic crystal fibre (PCF). Results are presented using two different types of polymer optical fibre (POF); first multimode PCF with a core diameter of 50µm based on poly(methyl methacrylate) (PMMA) and second, endlessly single mode PCF with a core diameter of 6µm based on TOPAS cyclic olefin copolymer. Bragg grating inscription was achieved using a 30mW continuous wave 325nm helium cadmium laser. Both TOPAS and PMMA fibre have a large attenuation of around 1dB/cm in the 1550nm spectral region, limiting fibre lengths to no longer than 10cm. However, both have improved attenuation of under 10dB/m in the 800nm spectral region, thus allowing for fibre lengths to be much longer. The focus of current research is to utilise the increased fibre length, widening the range of sensor applications. The Bragg wavelength shift of a grating fabricated in PMMA fibre at 827nm has been monitored whilst the POF is thermally annealed at 80°C for 7 hours. The large length of POF enables real time monitoring of the grating, which demonstrates a permanent negative Bragg wavelength shift of 24nm during the 7 hours. This creates the possibility to manufacture multiplexed Bragg sensors in POF using a single phase mask in the UV inscription manufacturing. TOPAS holds certain advantages over PMMA including a much lower affinity for water, this should allow for the elimination of cross-sensitivity to humidity when monitoring temperature changes or axial strain, which is a significant concern when using PMMA fibre.

Strategic marketing and business performance:a study in three European 'engineering countries'

In spite of its relevance, the effects of strategic marketing on business performance are sparingly studied, especially in particular business contexts. We address this gap in two ways. First, we examine the influence of four key strategic marketing concepts—market orientation, innovation orientation, and two marketing capability categories (outside-in and inside-out capabilities)—on company performance. Second, these relationships are studied in three European “engineering countries:” Austria, Finland and Germany. Their relative homogeneity enables testing the generality versus context-specificity of strategic marketing's performance impact. Using SEM analysis, surprisingly weak relationships between market orientation and outside-in capabilities, and business performance are identified, as opposed to the strong role of inside-out capabilities and innovation orientation. These results can be understood through the “engineering country” characteristics. Moreover, clear differences in results are identified among these relatively homogenous countries. This is a major finding as it challenges the widely assumed generality of the strategic marketing–performance relationship. Country-specific results have also considerable managerial relevance.

A cross-national investigation into the marketing department's influence within the firm:toward initial empirical generalizations

An ongoing strong debate within the marketing discipline concerns the role of marketing within the firm. It has been frequently reported that the marketing function is in a deep decline. Marketing executives and academics alike are interested in the antecedents of this decline and potential performance consequences of this decline. Recent academic research have started investigations on this important topic. Using studies in single countries innovativeness and accountability of the marketing department has been reported as major antecedents of the influence of the marketing department within the organization. Academic research, however, does not provide convincing evidence for a direct link between this influence and business performance. Instead it shows that market orientation is a crucial intervening variable, as marketing department influence is positively related market orientation, which subsequently positively related to business performance. As noted prior research, however, studies firms in single countries. In this article we execute a cross-national study on the antecedents and performance consequences of marketing department influence in order to derive initial empirical generalizations. This study is executed in seven Western-oriented countries, including USA, UK, The Netherlands, Germany, Sweden, Israel and Australia. The study heavily builds on the framework developed in the 2009 Journal of Marketing article of Verhoef and Leeflang. This framework is tested per country and subsequently meta-analytic tests are used to derive initial empirical generalizations. An important empirical generalization is that innovativeness, the customer-connecting capabilities, and accountability of the marketing department are positively related to marketing department influence. Interestingly, a second initial generalization is that creativity of the marketing negative induces less influence. Our results also show a third empirical generalization in that firms having a CEO with a marketing background tend to have more influential marketing departments. Confirming prior research a fourth initial empirical generalization is that MD influence measures and market orientation are positively related. Market orientation is subsequently positively related to business performance. Our most important generalization is, however, that MD influence is positively related to business performance. Hence, beyond striving to become market oriented, firms should also aim to have strong marketing departments. These departments can create a stronger focus on the customer and can also coordinate marketing efforts. In order to become more influential marketing departments should: (1) acquire innovative capabilities, (2) be more connected to customers, (3) invest in accountability, and (4) be careful with be careful being too creative.

A cross-national investigation into the marketing department's influence within the firm

Both marketing academics and practitioners are debating the diminished role of marketing as a separate function within firms. In this study, which expands on previous research on Dutch companies, the authors focus on how the marketing department’s capabilities relate to business performance across countries. The authors collected data in seven Western countries—the Netherlands, Germany, Sweden, United Kingdom, United States, Australia, and Israel. They surveyed top marketing and financial executives, CEOs, and other top employees of profit-based middle-sized and large firms. Their findings show that accountability provides the most consistent predictor of influence, whereas the marketing department’s innovativeness and customer connection show less consistent results. Across the seven countries, the department’s integration with the finance department has a consistent but negative effect on the department’s perceived influence. The influences of marketing departments clearly differ across countries. Perceived influence is substantially higher in the United States and Israel than in other countries, whereas top management respect for the marketing department is substantially higher in Israel than in any other country. The study also found that the marketing department is well represented on the boards of companies in Sweden, Israel, and the United States. In most countries, marketing tends not to be organized as a line function. Some differences among countries emerge in the relationships between the marketing department’s influence and business performance. In Israel, the United States, the United Kingdom, Germany, and Australia, influence relates positively to business performance, whereas in the Netherlands, it has no influence. The results for Sweden suggest a negative influence. The authors conclude that a strong marketing department appears to benefit firms in most of the countries studied. The results imply that the marketing department should have input into boardroom considerations.

Engineering of a novel Saccharomyces cerevisiae wine strain with a respiratory phenotype at high external glucose concentrations

The recently described respiratory strain Saccharomyces cerevisiae KOY.TM6*P is, to our knowledge, the only reported strain of S. cerevisiae which completely redirects the flux of glucose from ethanol fermentation to respiration, even at high external glucose concentrations (27). In the KOY.TM6*P strain, portions of the genes encoding the predominant hexose transporter proteins, Hxt1 and Hxt7, were fused within the regions encoding transmembrane (TM) domain 6. The resulting chimeric gene, TM6*. encoded a chimera composed of the amino-terminal half of Hxt1 and the carboxy-terminal half of Hxt7. It was subsequently integrated into the genome of an hxt null strain. In this study, we have demonstrated the transferability of this respiratory phenotype to the V5 hxt1-7Δ strain, a derivative of a strain used in enology. We also show by using this mutant that it is not necessary to transform a complete hxt null strain with the TM6* construct to obtain a nonethanol-producing phenotype. The resulting V5.TM6*P strain, obtained by transformation of the V5 hxt1-7Δ strain with the TM6* chimeric gene, produced only minor amounts of ethanol when cultured on external glucose concentrations as high as 5%. Despite the fact that glucose flux was reduced to 30% in the V5.TM6*P strain compared with that of its parental strain, the V5.TM6*P strain produced biomass at a specific rate as high as 85% that of the V5 wild-type strain. Even more relevant for the potential use of such a strain for the production of heterologous proteins and also of low-alcohol beverages is the observation that the biomass yield increased 50% with the mutant compared to its parental strain. Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Simple room temperature method for polymer optical fibre cleaving

In this paper, we report on a new method to cleave polymer optical fibre. The most common way to cut a polymer optical fibre is chopping it with a razor blade; however, in this approach both the fibre and the blade must be preheated in order to turn the material ductile, and thus, prevent crazing. In this paper, we make use of the temperature-time equivalence in polymers to replace the use of heating by an increase of the cleaving time and use a sawing motion to reduce fibre end face damage. In this way, the polymer fibre can be cleaved at room temperature in seconds with the resulting end face being of similar quality to those produced by more complex and expensive heated systems.