Genetically controlled cell lysis in the yeast Saccharomyces cerevisiae.

The cell wall of the yeast Saccharomyces cerevisiae is a tough, rigid structure, which presents a significant barrier to the release of native or recombinant proteins from this biotechnologically important organism. There is hence a need to develop inexpensive and efficient methods of lysing yeast cells in order to release their intracellular contents. To develop such a method, a tightly regulated promoter, pMET3, has been used to control three genes involved in cell wall biogenesis: PDE2, SRB1/PSA1, and PKC1. Two of these regulation cassettes, pMET3-SRB1/PSA1 and pMET3-PKC1, have been integrated at the chromosomal loci of the respective genes in order to overcome problems of plasmid instability. Although repression of PDE2 did not cause cell lysis, cells depleted of Srb1p/Psa1p gradually lost their viability and integrity, releasing about 10% of total protein into the medium. Repression of PKC1 led to extensive cell lysis, accompanied by the release of 45% of cellular protein into the medium. A double mutant, carrying both pMET3-SRB1/PSA1 and pMET3-PKC1 cassettes in place of SRB1/PSA1 and PKC1, was constructed and found to permit the efficient release of both homologous and heterologous proteins. © 1999 John Wiley & Sons, Inc.,     

Genetically engineered Pichia pastoris yeast for conversion of glucose to xylitol by a single-fermentation process

Xylitol is industrially synthesized by chemical reduction of d-xylose, which is more expensive than glucose. Thus, there is a growing interest in the production of xylitol from a readily available and much cheaper substrate, such as glucose. The commonly used yeast Pichia pastoris strain GS115 was shown to produce d-arabitol from glucose, and the derivative strain GS225 was obtained to produce twice amount of d-arabitol than GS115 by adaptive evolution during repetitive growth in hyperosmotic medium. We cloned the d-xylulose-forming d-arabitol dehydrogenase (DalD) gene from Klebsiella pneumoniae and the xylitol dehydrogenase (XDH) gene from Gluconobacter oxydans. Recombinant P. pastoris GS225 strains with the DalD gene only or with both DalD and XDH genes could produce xylitol from glucose in a single-fermentation process. Three-liter jar fermentation results showed that recombinant P. pastoris cells with both DalD and XDH converted glucose to xylitol with the highest yield of 0.078 g xylitol/g glucose and productivity of 0.29 g xylitol/L h. This was the first report to convert xylitol from glucose by the pathway of glucose–d-arabitol–d-xylulose–xylitol in a single process. The recombinant yeast could be used as a yeast cell factory and has the potential to produce xylitol from glucose.     

Genetically engineered wine yeast produces a high concentration of L-lactic acid of extremely high optical purity

For mass production of lactic acid, we newly constructed a transgenic wine yeast strain that included six copies of the bovine L-lactate dehydrogenase gene on the genome. On fermentation in inexpensive cane juice-based medium, L-lactate production of this recombinant reached 122 g/liter and the optical purity was 99.9% or higher.     

Genetically controlled IgM hyporesponsiveness to aK. pneumoniae polysaccharide

Examination of the immune response to aKlebsiella pneumoniae polysaccharide (K47-PS) has revealed that BALB/c mice demonstrate only a very weak primary response to this antigen. The low response does not result from either a peculiar dose response curve for BALB/c mice or from differing optimal antigen concentrations for high and low responder mice. Genetic analysis indicates that this variability of response is explicable assuming two alleles at a single locus; high responsiveness is dominant. Variability of response is probably not linked to theH-2 complex since the low and high responder mice, BALB/c and B10.D2/Sn new line, respectively, share the sameH-2 haplotype (H- 2_d). Tests of F₂s, backcrosses, and appropriate congenics have not shown evidence of linkage to sex, the albinism gene, the genes controlling coat color (agouti, black, brown), or the allotype-constant-region genes. The hyporesponsiveness is apparent only in the primary (IgM) response; hyperimmunization evokes similar antibody titers in high and low responding strains.     

Creation of cell surface-engineered yeast that display different fluorescent proteins in response to the glucose concentration

We have successfully created a novel yeast strain able to monitor changes in environmental conditions by displaying either green fluorescent protein (GFP) from Aequorea victoria or blue fluorescent protein (BFP), a variant of GFP, on its cell surface as a visible reporter. For the display of these fluorescent proteins on the cell surface of Saccharomyces cerevisiase, our cell-surface-engineering system was utilized. The GAPDH promoter, which is active in the presence of glucose, and the UPR-ICL promoter from Candida tropicalis, which starts to function in the presence of a reduced level of glucose, were employed simultaneously to express the GFP-encoding gene and the BFP-encoding gene, respectively. This cell-surface-engineered yeast strain emitted green fluorescence from the cell surface when sufficient glucose was present in the medium, and blue fluorescence from the same cell surface when the glucose in the medium was consumed. The fluorescent proteins displayed on the cell surface using the different promoters enabled us to monitor the concentrations of intra- and/or extracellular glucose that regulated activation or inactivation of the promoters. This novel yeast strain could facilitate the computerized control of various bioprocesses measuring emitted fluorescence.     

Multi-level response of the yeast genome to glucose

The yeast Saccharomyces cerevisiae shows a great variety of cellular responses to glucose via several glucose-sensing and signaling pathways. Recent microarray analysis has revealed multiple levels of genomic sensitivity to glucose and highlighted the power of genome-wide analysis to detect cellular responses to minute environmental changes.     

Genetically controlled anthocyanin synthesis in cell cultures of Matthiola incana

Callus cultures were derived from different parts of 8 anthocyanin producing and 2 white flowering lines of the crucifer Matthiola incana. The tissue cultures of the cyanic lines were shown to produce genotype specific anthocyanin patterns, whereas in the calli of the acyanic lines no anthocyanin synthesis occured.Abbreviations IAA indoleacetic acid - 2,4-D dichlorophenoxyacetic acid - MeOH methanol - Et₂O ether - ETOAc ethylacetate     

Mediated amperometry reveals two distinct modes of yeast responses to glucose

Mediated amperometry was exploited to monitor intracellular redox activity without cell disruption. Continuous measurements of menadione-mediated glucose currents at carbon paste electrodes with various immobilized intact wild type yeasts (Saccharomyces cerevisiae, Candida pulcherrima, Clavispora lusitaniae, Wickerhamomyces anomalus, Pichia guilliermondii, Kluyveromyces lactis var. lactis, Debaryomyces hansenii, Candida zeymolaydes and Candida tropicalis) revealed two distinct and previously unreported modes of development of the currents during the first 2 to 3 min. after subjection to glucose. A correlation among the values of the currents and the capacities of wild type yeasts to secrete various substances was observed.     

Mechanism of rapid-phase insulin response to elevation of portal glucose concentration

The hepatoportal region is important for glucose sensing; however, the relationship between the hepatoportal glucose-sensing system and the postprandial rapid phase of the insulin response has been unclear. We examined whether a rapid-phase insulin response to low amounts of intraportal glucose infusion would occur, compared that with the response to intrajugular glucose infusion in conscious rats, and assessed whether this sensing system was associated with autonomic nerve activity. The increases in plasma glucose concentration did not differ between the two infusions at 3 min, but the rapid-phase insulin response was detected only in the intraportal infusion. A sharp and rapid insulin response was observed at 3 min after intraportal infusion of a small amount of glucose but not after intrajugular infusion. Furthermore, this insulin response was also induced by intraportal fructose infusion but not by nonmetabolizable sugars. The rapid-phase insulin response at 3 min during intraportal infusion did not differ between rats that had undergone hepatic vagotomy or chemical sympathectomy with 6-hydroxydopamine compared with control rats, but this response disappeared in rats that had undergone chemical vagotomy with atropine. We conclude that the elevation of glucose concentration in the hepatoportal region induced afferent signals from undetectable sensors and that these signals stimulate pancreas to induce the rapid-phase insulin response via cholinergic nerve action.     

Genetically controlled quantitative variation of ornithine transcarbamylase in the chick kidney

This experiment was made to show that the marked variation in ornithine transcarbamylase (OTC) activity observed within a chicken breed or among breeds is due to quantitative changes, not qualitative ones. The enzyme was partially purified from three different chicken breeds, the White Leghorn B line, the Cochin Bantam breed, and a commercial line named "G," by the following steps: (i) extraction of OTC with Triton X-100 and cetyl-trimethylammonium bromide, (ii) heating, and (iii) salting-out column chromatography. No difference was shown immunologically, enzymologically, or physicochemically among the partially purified OTCs. The enzyme amount determined using anti-bovine OTC antiserum was related linearly to the enzyme activity either from the same chicken breed or from different breeds. These results suggest that marked variation in OTC activity reflects variation in the amount of enzyme synthesized in the kidney, and this is controlled by regulatory genes encoded on an autosome, not the structural gene.     

A yeast biosensor for glucose determination

Summary A yeast potentiometric biosensor for glucose determination is described. After induction of glycolytic enzyme synthesis a cell suspension of the yeast Hansenula anomala is retained in calcium alginate gel on the surface of a glass electrode. This biosensor gives a Nernstian response in glucose concentration of 5·10^–4–5·10^–3 mol/l with a response time of 5 min and a life-time of at least 2 months. Mannose and fructose are the only significantly interfering substances. The biosensor was used for measurement of glucose concentration in urine with results comparable to those obtained by a photometric enzymatic method.     

The glucose signaling network in yeast

Background

Most cells possess a sophisticated mechanism for sensing glucose and responding to it appropriately. Glucose sensing and signaling in the budding yeast Saccharomyces cerevisiae represent an important paradigm for understanding how extracellular signals lead to changes in the gene expression program in eukaryotes.

Scope of review

This review focuses on the yeast glucose sensing and signaling pathways that operate in a highly regulated and cooperative manner to bring about glucose-induction of HXT gene expression.

Major conclusions

The yeast cells possess a family of glucose transporters (HXTs), with different kinetic properties. They employ three major glucose signaling pathways—Rgt2/Snf3, AMPK, and cAMP-PKA—to express only those transporters best suited for the amounts of glucose available. We discuss the current understanding of how these pathways are integrated into a regulatory network to ensure efficient uptake and utilization of glucose.

General significance

Elucidating the role of multiple glucose signals and pathways involved in glucose uptake and metabolism in yeast may reveal the molecular basis of glucose homeostasis in humans, especially under pathological conditions, such as hyperglycemia in diabetics and the elevated rate of glycolysis observed in many solid tumors.     

Role of glucose signaling in yeast metabolism

In this article, knowledge concerning the relation between uptake of and signaling by glucose in the yeast Saccharomyces cerevisiae is reviewed and compared to the analogous process in prokaryotes. It is concluded that (much) more fundamental knowledge concerning these processes is required before rational redesign of metabolic fluxes from glucose in yeast can be achieved. (c) 1996 John Wiley & Sons, Inc.     

Nicotine-evoked conditioned responding is dependent on concentration of sucrose unconditioned stimulus

Previous studies have shown that the interoceptive nicotine conditional stimulus (CS) functions similarly to exteroceptive CSs such as lights or environments. For instance, the appetitive conditioned response (CR) evoked when nicotine is repeatedly paired with sucrose presentations (the unconditioned stimulus; US) is sensitive to changes in training dose (CS salience) and the contiguity between the CS effects and sucrose. The current study was conducted to extend this research by examining the possible role of US intensity in CR acquisition and maintenance. Rats were trained using one of four sucrose concentrations: 0, 4, 16, or 32% (w/v). On nicotine sessions (0.4 mg base/kg), rats received 36 deliveries (4 s each) of their assigned concentration intermittently throughout the session; sucrose was withheld on saline sessions. In all groups, an appetitive goal-tracking CR was acquired at a similar rate. However, the asymptotic CR level varied with sucrose concentration. The magnitude of the CR was increased in rats trained with higher sucrose US concentrations. These findings are consistent with previous Pavlovian conditioning research, and extend the conditions under which the nicotine state functions as an interoceptive conditional stimulus.     

Yeast galactose permease is related to yeast and mammalian glucose transporters

We have cloned and sequenced the GAL2 gene of Saccharomyces cerevisiae, which encodes galactose permease. The GAL2 protein is related to the yeast glucose transporter encoded by the SNF3 gene, and also to mammalian and bacterial sugar permeases. Like the other members of this protein family, GAL2 has twelve hydrophobic segments that are separated by loops of charged amino acids. A comparison of different members of this protein family shows that those parts of the polypeptides thought to be on the cytoplasmic side of the cell membrane, are more conserved than other parts of the molecules.