Multiplicity of mechanisms of carbon catabolite repression involved in the synthesis of alcohol oxidase in the methylotrophic yeast Pichia pinus

The effect of various carbon compounds on the synthesis of alcohol oxidase in a medium with methanol was studied in the wild type strain of Pichia pinus as well as in gcr1 and ecr1 mutants defective in glucose and ethanol repression of methanol metabolic enzymes, respectively. Compounds repressing the synthesis of alcohol oxidase in the wild type strain were divided into four groups. Repression of alcohol oxidase by compounds of the first group (glucose, fructose, mannose, galactose, L -sorbose and xylose) was impaired only in the gcr1 mutant and that by compounds of the second group (ethanol, acetate, 2-oxoglutarate and erythritol) only in the ecr1 mutant. Repression by compounds of the third group (malate, dihydroxyacetone) was not impaired in both these regulatory mutants and that by compounds of the fourth group (succinate, fumarate, L -arabinose, sorbitol, salicin, xylitol and cellobiose) was partially reduced in both gcr1 and ecr1 strains. Mutation gcr1 causes a significant decrease in phosphofructokinase activity. It also led to a six- to seven-fold increase in intracellular pools of glucose-6-phosphate and fructose-6-phosphate and to a two-fold decrase in the intracellular pool of fructose-1,6-bisphosphate. In ecr1 strains, a decrese in 2-oxoglutarate dehydrogenase activity accompanied by an increae in activities of NAD- and NADP-dependent isocitrate dehydrogenases and NAD- and NADP-dependent glutamate dehydrogenases was demonstrated. The intracellular pool of 2-oxoglutarate was increased 2·5-fold in ecr1 strains. Genes GCR1 and ECR1 are not linked. The mechanisms of catabolite repression of alcohol oxidase in methylotrophic yeasts are discussed.     

Effect of dimorphic regulation on heterologous glucose oxidase production by Mucor circinelloides

The production of Aspergillus niger glucose oxidase (GOX) and native amylase by the recombinant M. circinelloides KFA199 strain under conditions of dimorphic growth was investigated. The recombinant KFA199 strain was compared to its parental ATCC 1216b strain and a wild‐type CBS 232.29 strain under similar morphology‐controlled conditions. Cultivation in Vogel's medium supplemented with ergosterol/Tween‐80 and sparged with nitrogen gas was most suitable for yeast‐like biomass production under anaerobic conditions. Anaerobic growth was characterized by high levels of ethanol formation and linear growth rates of 0.24–0.05/h, indicating metabolic stress. Subsequent to anaerobic growth, cultures were shifted to aerobic conditions to induce aerobic mycelial growth. GOX produced by the recombinant KFA199 after the shift to aerobic conditions was poorly secreted and accumulated intracellularly to 0.56 U/ml_culture. Amylase production by the KFA199, ATCC12b and CBS 232.29 strains was determined during growth on starch after the shift to aerobic culture. Growth‐associated amylase production by the ATCC 1216b (0.63 U/ml_culture) and wild‐type CBS 232.29 (0.33 U/ml_culture) strains was substantially higher than by the recombinant KFA199 strain (0.07 U/ml_culture), which may be related to the leucine auxotrophy of the transformation host, or genetic changes induced during transformation of the KFA199 strain. Copyright © 2010 John Wiley & Sons, Ltd.     

Antibacterial Effect of Glucose Oxidase on Growth of Pseudomonas fragi as Related to pH

The effect of glucose oxidase (GOX) catalyzed reaction with glucose on Pseudomonas fragi was analyzed in nutrient broth and fish extract media. Growth of P. fiugi in nutrient broth was clearly suppressed by 1.0, 2.0 and 4.0 mg/mL glucose when combined with 0.5–2.0 U/mL GOX. The same GOX/glucose combinations inhibited P. frasi growth in fish extract media. Viable cell numbers in fish media showed clear growth inhibition with combinations of l.0–2.0 U/mL GOX and 8.0–16.0 mg/mL glucose. Higher GOX and glucose rapidly produced 2.0–2.5 unit decreases in pH, but produced enough gluconic acid to precipitate fish proteins. Use of 0.5 U/mL GOX in fish extract media resulted in slow, sustained activity with potential for inhibition of microbial growth in foods without excessive acidity.     

Partial biochemical characterization of tetrazolium red‐reactive Lactobacillus plantarum mutants

One thousand colonies derived from Lactobacillus plantarum ATCC 8014 cells that survived 34–43 × 10³ ergs cm^‐2 ultraviolet irradiation were screened on media containing tetrazolium red to detect fermentative mutants. Fermentation end‐products formed from pyruvate, glucose, or lactose catabolism were determined. All 37 stable tetrazolium red‐reactive mutants had increased pyruvate utilization compared to the wild‐type strain. Only two did not produce lactate from exogenous pyruvate. When glucose or lactose were substrates, these two mutants and six other representative mutants produced lactate at levels similar to the parent strain. Although the average lactate and acetoin production from pyruvate by the mutants and wild‐type strains were similar, 25% of the mutants had increased acetoin production.     

GLUCOSE OXIDASE REACTION FOR ESTIMATION OF GLUCOSE WITHOUT HORSERADISH PEROXIDASE. SOME MICROBIOLOGICAL AND FERMENTATION APPLICATIONS

An enzymic method has been developed for analysis of glucose. Glucose oxidase acts on glucose to produce hydrogen peroxide which acts to directly reduce the green Cu(II) 2–2′-bicinchoninate complex to a violet complex without horseradish peroxidase. A concentration range of 20–200 μM glucose was used but the reaction shows a linear range of 20–800 μM glucose. Interference is controlled by using a blank determination which has not been treated with glucose oxidase. The reaction has been used to estimate glucose levels in Saccharomyces cerevisiae fermentation and α-amylase, invertase and β-galactosidase reactions and coloured corn-steep fermentation media.     

High-affinity glucose uptake in Saccharomyces cerevisiae is not dependent on the presence of glucose-phosphorylating enzymes

Glucose uptake in Saccharomyces cerevisiae is believed to consist of two kinetically distinguishable components, the affinity of which is modulated during growth on glucose. It has been reported that triple hexose-kinase deletion mutants do not exhibit high-affinity glucose uptake. This raises the question of whether and how high-affinity glucose uptake is related to the presence of glucose-phosphorylating enzymes. In this study the kinetics of glucose uptake in both wild-type cells and cells of hexose-kinase deletion mutants, grown on either glycerol or galactose, were determined using a rapid-uptake method. In wild-type cells glucose uptake measured over either 5 s or 200 ms exhibited high affinity. In contrast, in cells of hexose-kinase deletion mutants the apparent affinity of glucose uptake was dependent on the time scale during which uptake was measured. Measurements on the 5-s scale showed apparent low-affinity uptake whereas measurements on the 200-ms scale showed high-affinity uptake. The affinity and maximal rate of the latter were comparable to those in wild-type cells. Using a simple model for a symmetrical facilitator, it was possible to simulate the experimentally determined relation between apparent affinity and the time scale used. The results suggest that high-affinity glucose transport is not necessarily dependent on the presence of glucose-phosphorylating enzymes. Apparent low-affinity uptake kinetics can arise as a consequence of an insufficient rate of removal of intracellular free glucose by phosphorylation. This study underlines the need to differentiate between influences of the translocator and of metabolism on the apparent kinetics of sugar uptake in yeast.     

Modification of Cellulomonas flavigena NTOU 1 Characteristics for the Production of Shrimp Hydrolysates

Two mutants (Type A and B) induced from Cellulomonas flavigena NTOU 1 demonstrated higher casein-hydrolyzing activity than mother culture. The optimum pH for hydrolyzing casein (pH 8–10) of the mutants and wild type mother culture strain was higher than that for growth (pH 6–7.5). When three strains were employed to hydrolyze whole shrimp, mutant A showed the highest yield (63.5%) of shrimp hydrolysate (SH), followed by mutant B (58.6%) and wild type culture (50.0%). Mutant B produced the best quality of SH and was considered safe, according to animal test, as a starter culture for shrimp sauce processing.     

Further Characterization of a Caffeine-Resistant Mutant of Aspergillus parasiticus

Studies were performed to characterize further Aspergillus parasiticus BCR1, a caffeine-resistant mutant of A. parasiticus NRRL 2999, particularly in regard to its caffeine-dependent production of aflatoxins. The enhanced synthesis of aflatoxins by caffeine was highly specific since neither dimethylxanthines nor purines could substitute for the trimethylxanthine. Caffeine's effects were phase dependent and only increased toxin formation if added early in the microorganism's life cycle. The ability of BCR1 to exclude caffeine appeared dependent on the initial levels of caffeine in the growth medium. Respiration and glucose utilization in the wild type strain were inhibited strongly by caffeine, but BCR1 was resistant to these effects. Comparison of glucose uptake kinetics in the wild type and mutant strains indicated that caffeine inhibition of aflatoxin synthesis in the wild type was not due to a disruption of glucose transport.     

Restoration of peroxisome formation in two conditional peroxisome-deficient mutants of Hansenula polymorpha during growth of cells on specific organic nitrogen sources

Expression of the peroxisome-deficient (Per^?) phenotype by per mutants of Hansenula polymorpha is shown to be dependent on specific environmental conditions. Analysis of our collection of constitutive and conditional per mutants showed that, irrespective of the carbon source used, the mutants invariably lacked functional peroxisomes when ammonium sulphate was used as a nitrogen source. However, in two temperature-sensitive (ts) mutants, per13-6^ts and per14-11^ts, peroxisomes were present at the restrictive temperature when cells were grown on organic nitrogen sources which are known to induce peroxisomes in wild-type cells, namely D -alanine (for both mutants) or methylamine (for per14-11^ts). These organelles displayed normal wild-type properties with respect to morphology, mode of development and protein composition. However, under these conditions not all the peroxisomal matrix proteins synthesized were correctly located inside peroxisomes. Detailed biochemical and (immuno) cytochemical analyses indicated that during growth of cells on methanol in the presence of either D -alanine or methylamine, a minor portion of these proteins (predominantly alcohol oxidase, dihydroxyacetone synthase and catalase) still resided in the cytosol. This residual cytosolic activity may explain the observation that the functional restoration of the two ts mutants is not complete under these conditions, as is reflected by the retarded growth of the cells in batch cultures on methanol.     

Cell wall structure suitable for surface display of proteins in Saccharomyces cerevisiae

A display system for adding new protein functions to the cell surfaces of microorganisms has been developed, and applications of the system to various fields have been proposed. With the aim of constructing a cell surface environment suitable for protein display in Saccharomyces cerevisiae, the cell surface structures of cell wall mutants were investigated. Four cell wall mutant strains were selected by analyses using a GFP display system via a GPI anchor. β‐Glucosidase and endoglucanase II were displayed on the cell surface in the four mutants, and their activities were evaluated. mnn2 deletion strain exhibited the highest activity for both the enzymes. In particular, endoglucanase II activity using carboxymethylcellulose as a substrate in the mutant strain was 1.9‐fold higher than that of the wild‐type strain. In addition, the activity of endoglucanase II released from the mnn2 deletion strain by Zymolyase 20T treatment was higher than that from the wild‐type strain. The results of green fluorescent protein (GFP) and endoglucanase displays suggest that the amounts of enzyme displayed on the cell surface were increased by the mnn2 deletion. The enzyme activity of the mnn2 deletion strain was compared with that of the wild‐type strain. The relative value (mnn2 deletion mutant/wild‐type strain) of endoglucanase II activity using carboxymethylcellulose as a substrate was higher than that of β‐glucosidase activity using p‐nitrophenyl‐β‐glucopyranoside as a substrate, suggesting that the cell surface environment of the mnn2 deletion strain facilitates the binding of high‐molecular‐weight substrates to the active sites of the displayed enzymes. Copyright © 2014 John Wiley & Sons, Ltd.     

Liposome-mediated introduction of proteins into protoplasts of the yeast Hansenula polymorpha as a possible tool to study peroxisome biogenesis

Low pH-induced fusion between liposomes and protoplasts of the yeast Hansenula polymorpha was demonstrated using a fluorescent assay and freeze-etch techniques. By this method foreign proteins could be introduced into the cytosol of the protoplast. For instance, after fusion of glucose oxidase-containing liposomes with protoplasts, activity of this enzyme was demonstrated cytochemically in the cytosol of the resulting protoplasts. Similar results were obtained when ferritin-containing liposomes were used. Incorporation of foreign proteins into liposomes was not a prerequisite for their introduction into the cytosol of protoplasts. In experiments where ferritin was added to protoplast suspensions together with empty liposomes, this protein was also delivered to the protoplast cytosol. However, in the absence of liposomes no uptake of proteins occurred. We tested the potential of this system in our studies on peroxisome biogenesis. Protoplasts of glucose-grown H. polymorpha remained stable for prolonged periods in osmotically stabilized cultivation media. Peroxisomes in such protoplasts were capable of protein import and assembly of matrix proteins as was demonstrated by the 20-fold increase in catalase activity after incubation with methanol or ethanol. However, mature alcohol oxidase purified from H. polymorpha introduced into protoplasts of glucose-grown cells of this organism was not targeted to peroxisomes as demonstrated with (immuno)cytochemical techniques. The enzyme remained present in the cytosol while its activity gradually decreased. Therefore mature alcohol oxidase probably does not expose the right topogenic signal(s) for recognition by its target organelle.     

Mutants of the methylotrophic yeast Pichia pinus defective in C2 metabolism

A collection of mutants of Pichia pinus which are unable to grow on ethanol but retain the ability to grow on glucose and methanol, was obtained. Genetic and biochemical analysis of these strains revealed mutations in seven nuclear genes affecting activities of isocitrate lyase (icl1), malate synthase (mls1), phosphoenolpyruvate carboxykinase (pck1), ‘malic’ enzyme (mdd1) and acetyl-CoA synthetase (acs1, acs2 and acs3). All mutations except acs1-acs3 have no effect on the activities of other enzymes involved in C₂ metabolism. Mutations acs1, acs2 and acs3 have a pleiotropic action, leading to partial reduction in activities of isocitrate lyase and malate synthase. Ethanol-induced repression of the synthesis of the methanol oxidative enzymes, alcohol oxidase and catalase, is not impaired in these seven mutant classes. On the other hand, C₂ compound-induced inactivation of alcohol oxidase and catalase is impaired in mutants acs1, acs2, acs3 and icl1. It was suggested that glyoxylate and acetate (or acetate precursors) act as low molecular weight effectors, ‘switching on’ inactivation and repression, respectively, of alcohol oxidase and catalase in the medium containing ethanol or acetate.     

Inhibition of food-related pathogenic bacteria by god-transformed Penicillium nalgiovense strains.

Two strains of Penicillium nalgiovense, which carried the god gene of Aspergillus niger and had increased glucose oxidase (GOD) activity compared with the wild-type strain, were tested for their ability to suppress the growth of certain food-related pathogenic bacteria. In contrast to the wild type, which showed no antibacterial effect when grown in mixed culture with different bacteria, the two transformed strains were highly antagonistic. The strain that expressed higher amounts of GOD in general had higher inhibitory activity. Both strains showed antibacterial activity against Listeria monocytogenes, Salmonella Enteritidis, and Staphylococcus aureus. The inhibitory activity was dependent on the glucose concentration in the medium. S. aureus was completely inhibited at 1% glucose in the presence of the higher GOD-producing transformant. In contrast, if arabinose was used as a carbon source, no inhibition occurred. If catalase was added to the medium, the inhibitory activity of the transformants was completely inactivated, indicating that the hydrogen peroxide produced was responsible for the antibacterial activity of the transformants.     

用于酶热传感器的酶标甲胺磷农药的制备

采用碳二亚胺(EDC)法进行酶标甲胺磷的制备研究。考察甲胺磷和丁二酸酐比例、反应时间对甲胺磷衍生物合成的影响,最终确定二者的最适物质的量比为1:1.5,最适反应时间为15h;甲胺磷衍生物与葡萄糖氧化酶比例对酶标甲胺磷偶联率及酶活的影响实验结果显示,随着甲胺磷衍生物与葡萄糖氧化酶二者比例的增加,酶标农药的偶联率增加,但同时也造成了酶活的降低。考虑到酶标农药还应具备足够的酯酶抑制活性,甲胺磷衍生物与葡萄糖氧化酶二者物质的量比为10:1时制得的酶标农药为最优。     

The SKS1 protein kinase is a multicopy suppressor of the snf3 mutation of Saccharomyces cerevisiae

Saccharomyces cerevisiae strains carrying snf3 are defective in high affinity glucose transport, and thus are unable to grow fermentatively on media with low concentrations of glucose. A multicopy suppressor of the snf3 growth defect, SKS1 (suppressor kinase of snf3), was found to encode a putative ser/thr protein kinase homologous to Ran1p, a kinase that regulates the switch between meiosis and vegetative growth in Schizosaccharomyces pombe. Overexpression of the SKS1 open reading frame is sufficient for suppression of the growth defects of snf3 mutants. Disruption of the open reading frame eliminates this suppression; as does the mutation of the consensus ATP binding site of Sks1p. A DDSE (DNA dependent snf3 suppressor element) was found to be present in the SKS1 promoter region. The suppression by this DDSE occurs in the absence of SKS1 coding region, that is, the DDSE can suppress a snf3 sks1 double null mutant which fails to grow fermentatively on low glucose as a snf3 mutant does. Both SKS1 and its DDSE can additionally suppress the growth defects of grr1 mutants, which are also impaired in high affinity glucose transport. The snf3 genomic suppressors, rgt1, RGT2 and ssn6, are also capable of suppressing snf3 associated growth defects in a strain lacking sks1.     

STUDIES ON CONJUGATE EFFECTS OF SUBSTRATE (GLUCOSE) AND PRODUCT (ETHANOL) ON CELL GROWTH KINETICS DURING FERMENTATION OF DIFFERENT YEAST STRAINS

The conjugate effect of substrate (glucose) and product (ethanol) has been studied on yeast growth during the course of fermentation. The study was made on five yeast strains Kloeckera apiculata, Saccharomyces uvarum, Saccharomyces bayanus, Saccharomyces cerevisiae UG5, and Saccharomyces cerevisiae sake. The results attest a different action of added alcohol as compared with produced alcohol for all strains. It is also verified that kinetic behaviour, namely ethanol inhibition, varies with initial substrate concentration, and so a new parameter, the added alcohol concentration at which biomass productivity declines to one-half its value with no added ethanol, is proposed to quantify ethanol inhibition     

The C-terminal Domain of Snf3p is Sufficient to Complement the Growth Defect of snf3 Null Mutations in Saccharomyces cerevisiae: SNF3 Functions in Glucose Recognition

The SNF3 protein, Snf3p, of Saccharomyces cerevisiae was initially thought to be a high affinity glucose transporter required for efficient catabolism of low glucose concentrations. We now report evidence suggesting that Snf3p is a regulatory protein and not a catabolic transporter. The C-terminal domain of Snf3p is able to complement the growth defect on solid media of snf3 null mutants independent of attachment to the membrane-spanning domains. However, the C-terminal domain is unable to fully restore high affinity glucose transport to a snf3 null strain. Examination of deletions of the C-terminal domain of intact SNF3 demonstrates that this region is required for both the growth and transport functions of Snf3p. Loss of the SNF3 gene leads to a long-term adaptation phenotype for cells grown in liquid medium at low substrate concentrations in the presence of the respiratory inhibitor, antimycin A. The presence of the C-terminal domain shortens the time required for adaptation in a snf3 null strain. Thus, Snf3p appears to affect ability to adapt to low substrate conditions, but does not confer an absolute defect in uptake of substrate. Taken together, these data suggest that Snf3p is a regulatory protein likely functioning in the detection of glucose. © 1997 by John Wiley & Sons, Ltd.     

产葡萄糖氧化酶菌株的诱变筛选及遗传稳定性研究

采用平板显色初筛、Underbofler滴定法复筛及紫外-硫酸二乙酯复合诱变的方法,从临沂果园土样中得到一株产葡萄糖氧化酶（GOD）活性较好的菌株,命名为Bla-6,其产GOD酶活为（10.50±0.25） U/mL,与原始菌株LPA-4产GOD酶活（6.75 U/mL）相比提高了55.57%。结合5.8S rDNA-ITS区序列系统进化、菌落形态、生长特性、菌体形态等确定其分类地位。结果表明,所得菌株Bla-6被鉴定为黑曲霉（Aspergillus niger）,经8代传代培养后,GOD酶活维持在10.5 U/mL左右,该菌株遗传稳定性较好。