Utilization of amines by yeasts

461 Strains of the yeast collection of the Centraalbureau voor Schimmelcultures (CBS) were screened for their ability to utilize 9 different amines as a sole carbon and energy source and/or as nitrogen source. A miniaturized technique with microtiter plates was used. None of the primary and methylated amines tested (i.e. methylamine, dimethylamine, trimethylamine, tetramethylammonium chloride, choline, ethylamine, propylamine, butylamine and benzylamine) were utilized as a carbon and energy source, although the majority of yeasts (86%) were able to utilize one or more of these compounds as a nitrogen source. The ability to utilize ethylamine and higher homologues occurred more frequently than the ability to utilize methylated amines. In almost all genera the utilization of primary and methylated amines was found, with utilizing and non-utilizing species occurring within a genus. The occurrence of specific assimilation patterns of amine utilization among yeasts and the inability of these organisms to utilize amines as a carbon and energy source is discussed.     

Cycloheximide resistance in the Lipomycetaceae revisited

Cycloheximide (CYH) is a heterocyclic, glutarimide antibiotic that is a potent inhibitor of protein biosynthesis in most eukaryotes. This study demonstrated that yeasts from all species of the Lipomycetaceae, with the exception of Dipodascopsis spp., can grow in the presence of up to 5 g.L(-1) CYH -- a concentration that is five times higher than the accepted "highest" concentration of 1 g.L(-1) used in physiological tests for yeast identification. Lipomycetaceous yeasts are known to utilize heterocyclic nitrogen-containing compounds such as thymine as sole nitrogen source. CYH contains a glutarimide ring, which is chemically similar to thymine. We investigated the possibility that CYH resistance in the Lipomycetaceae might be due to an ability to degrade CYH and use it as the sole nitrogen source. However, we were unable to demonstrate significant growth on CYH as sole nitrogen source. When thymine was used as positive control, we could demonstrate its utilization as sole nitrogen source.     

Genetic Basis of Variations in Nitrogen Source Utilization in Four Wine Commercial Yeast Strains

The capacity of wine yeast to utilize the nitrogen available in grape must directly correlates with the fermentation and growth rates of all wine yeast fermentation stages and is, thus, of critical importance for wine production. Here we precisely quantified the ability of low complexity nitrogen compounds to support fast, efficient and rapidly initiated growth of four commercially important wine strains. Nitrogen substrate abundance in grape must failed to correlate with the rate or the efficiency of nitrogen source utilization, but well predicted lag phase length. Thus, human domestication of yeast for grape must growth has had, at the most, a marginal impact on wine yeast growth rates and efficiencies, but may have left a surprising imprint on the time required to adjust metabolism from non growth to growth. Wine yeast nitrogen source utilization deviated from that of the lab strain experimentation, but also varied between wine strains. Each wine yeast lineage harbored nitrogen source utilization defects that were private to that strain. By a massive hemizygote analysis, we traced the genetic basis of the most glaring of these defects, near inability of the PDM wine strain to utilize methionine, as consequence of mutations in its ARO8, ADE5,7 and VBA3 alleles. We also identified candidate causative mutations in these genes. The methionine defect of PDM is potentially very interesting as the strain can, in some circumstances, overproduce foul tasting H₂S, a trait which likely stems from insufficient methionine catabolization. The poor adaptation of wine yeast to the grape must nitrogen environment, and the presence of defects in each lineage, open up wine strain optimization through biotechnological endeavors.     

Translocation of a discrete piece of deoxyribonucleic acid carrying an amp gene between replicons in Eschericha coli.

Uptake and intracellular transformation of pyrimidines supplying cells of the yeast Rhodotorula glutinis with nitrogen have been studied. The amine nitrogen of cytosine was found to be the easiest to utilize. The presence in the medium of inorganic ammonia along with cytosine had a slight effect on cytosine deaminase (EC 3.5.4.1) activity. The uracil produced entered into the nutrient medium with no fission break of the pyridmidine ring. In the absence of any other source of nitrogen, the cells of the yeast R. glutinis utilized nitrogen of the pyrimidine ring of oxypyrimidines. Catabolism of uracil followed the reductive pattern, with release of carbon dioxide; this was accompanied by synthesis of the key enzyme of pyrimidine catabolism, dihydrouracil dehydrogenase (EC 1.3.1.1), whose activity rose 10-fold. With thymidne as the sole source of nitrogen, the lag-phase growth of the yeast cells was maximum. Catabolism of the pyrimidine ring of thymine was possibly preceded by its transformation into uracil. With no source of nitrogen easily utilized, the uridine 5'-monophosphate content in the generally acid-soluble pool rose. Our discussion of the regulation of catabolism of exogenous pyrimidine bases by the yeast R. glutinis takes into account the fact that transformations of pyrimidine bases are determined by how easily the cells can use a particular base as a source of nitrogen.     

Occurrence of a novel yeast enzyme, L-lysine epsilon-dehydrogenase, which catalyses the first step of lysine catabolism in Candida albicans

The yeast Candida albicans is able to utilize L-lysine as the sole nitrogen and carbon source accompanied by intracellular accumulation of alpha-aminoadipate-delta-semialdehyde. A novel yeast amino acid dehydrogenase catalysing the oxidative deamination of the epsilon-group of L-lysine was found in this yeast. The enzyme, L-lysine epsilon-dehydrogenase, is strongly induced in cells grown on L-lysine as the sole nitrogen source. The enzyme is specific for both L-lysine and NADP+. The Km values were determined to be 0.87 mM for L-lysine and 0.071 mM for NADP+. An apparent Mr of 87,000 was estimated by gel filtration. The enzyme has maximum activity at pH 9.5 and a temperature optimum of 32 degrees C under our assay conditions.     

Theanine production by coupled fermentation with energy transfer employing Pseudomonas taetrolens Y-30 glutamine synthetase and baker's yeast cells

Theanine was formed from glutamic acid and ethylamine by coupling the reaction of glutamine synthetase (GS) of Pseudomonas taetrolens Y-30 with sugar fermentation of baker's yeast cells as an ATP-regeneration system. Theanine formation was stimulated by the addition of Mn2+ to the mixture for the coupling. The addition of Mg2+ was less effective. In a mixture containing a larger amount of yeast cells with a fixed level of GS, glucose (the energy source) was consumed rapidly, resulting in a decrease in the final yield of theanine. On the other hand, an increase in GS amounts increased theanine formation in a mixture with a fixed amount of yeast cells. High concentrations of ethylamine enhanced theanine formation whereas inhibited yeast fermentation of sugar and the two contrary effects of ethylamine caused a high yield of theanine based on glucose consumed. In an improved reaction mixture containing 200 mM sodium glutamate, 1,200 mM ethylamine, 300 mM glucose, 50 mM potassium phosphate buffer (pH 7.0), 5 mM MnCl2, 5 mM AMP, 100 units/ml GS, and 60 mg/ml yeast cells, approximately 170 mM theanine was formed in 48 h.     

Metabolism of the herbicide bromoxynil by Klebsiella pneumoniae subsp. ozaenae.

Enrichment of soil samples for organisms able to utilize the herbicide bromoxynil (3,5-dibromo-4-hydroxybenzonitrile) as a nitrogen source yielded bacterial isolates capable of rapidly metabolizing this compound. One isolate, identified as Klebsiella pneumoniae subsp. ozaenae, could completely convert 0.05% bromoxynil to 3,5-dibromo-4-hydroxybenzoic acid and use the liberated ammonia as a sole nitrogen source. Assays of cell extracts of this organism for the ability to produce ammonia from bromoxynil revealed the presence of a nitrilase (EC 3.5.51) activity. The enzyme could not utilize 3,5-dibromo-4-hydroxybenzamide as a substrate, and no 3,5-dibromo-4-hydroxybenzamide could be detected as a product of bromoxynil transformation. Comparison of related aromatic nitriles as substrates demonstrated that the Klebsiella enzyme is highly specific for bromoxynil.     

Metabolism of the herbicide bromoxynil by Klebsiella pneumoniae subsp. ozaenae

Enrichment of soil samples for organisms able to utilize the herbicide bromoxynil (3,5-dibromo-4-hydroxybenzonitrile) as a nitrogen source yielded bacterial isolates capable of rapidly metabolizing this compound. One isolate, identified as Klebsiella pneumoniae subsp. ozaenae, could completely convert 0.05% bromoxynil to 3,5-dibromo-4-hydroxybenzoic acid and use the liberated ammonia as a sole nitrogen source. Assays of cell extracts of this organism for the ability to produce ammonia from bromoxynil revealed the presence of a nitrilase (EC 3.5.51) activity. The enzyme could not utilize 3,5-dibromo-4-hydroxybenzamide as a substrate, and no 3,5-dibromo-4-hydroxybenzamide could be detected as a product of bromoxynil transformation. Comparison of related aromatic nitriles as substrates demonstrated that the Klebsiella enzyme is highly specific for bromoxynil.     

Pleiotropic mutants affecting the control of nitrogen metabolism in Aspergillus nidulans

Summary Mutants of Aspergillus nidulans with lesions in gene amdT are pleiotropically affected in their ability to utilize a wide variety of nitrogen sources in the presence of glucose. Ability to utilize a number of these compounds as sole sources of carbon and nitrogen is not altered. One of these mutants, amdT102, has properties consistent with it being derepressed for glucose repression of the utilization of most (but not all) nitrogen sources. The amdT102 mutant can grow strongly on histidine, lysine and cystine as sole nitrogen sources while the wild type strain grows extremely poorly on these amino acids. Similar but less extreme effects apply to many other nitrogen sources. The amdT19 mutant is unable to utilize most nitrogen sources in the presence of glucose, suggesting that it is subject to greatly increased repression of nitrogen source utilization. The amdT mutants are not affected in their ability to use many compounds as sole carbon sources. Carbon sources other than glucose also affect utilization of nitrogen sources in the amdT mutants.     

Regulation of phenylalanine ammonia lyase in Rhodotorula glutinis.

In the red yeast Rhodotorula glutinis, phenylalanine ammonia lyase (PAL) was induced 10-fold during carbon starvation even in the absence of exogenous phenylalanine, although maximal induction occurred when phenylalanine was the nitrogen (40-fold) or carbon (100-fold) source. Apparent regulatory mutations that affected the expression of PAL were isolated by selecting mutants resistant to the analog p-fluoro-D,L-phenylalanine (PFP). One such mutant, designated FP1, could use phenylalanine as a nitrogen source but not as a carbon source. Similarly, FP1 failed to utilize intermediates of the phenylalanine degradative pathway, namely, benzoate, p-hydroxybenzoate, or 3,4-dihydroxybenzoate, as carbon sources. Although the PFP-resistant mutant contained a low level of PAL, no increase was found when it was grown with phenylalanine as the nitrogen source. A derivative of FP1, FP1a, was isolated that simultaneously regained an inducible PAL and the ability to use phenylalanine, benzoate, p-hydroxybenzoate, and 3,4-dihydroxybenzoate as carbon sources. In addition, when p-hydroxybenzoate was the carbon source, PAL was induced in the mutant FP1a but not in the PFP-sensitive parental strain. We propose that the mutation to PFP resistance occurred in a regulatory gene that controls the entire phenylalanine degradative pathway. Secondary mutations at this locus, as found in strain FP1a, not only restored expression of this pathway, but also altered the induction of PAL by metabolites of this pathway.     

Theanine production by coupled fermentation with energy transfer using gamma-glutamylmethylamide synthetase of Methylovorus mays No. 9

Gamma-glutamylmetylamide synthetase (GMAS) of Methylovorus mays No. 9, produced by Eschericia coli AD494 (DE3) harboring pET21aGM, formed theanine from glutamic acid and ethylamine with coupling of the reaction with sugar fermentation of baker's yeast cells as an ATP-regeneration system. Theanine formation was stimulated by the addition of Mn(2+) to the reaction mixture, whereas Mg(2+) was less effective. Increases to a certain level in the concentrations of GMAS and the substrates in the mixture were effective in increasing theanine formation, but high concentrations of ethylamine (900 mM or more) inhibited yeast sugar fermentation, and eventually decreased theanine formation. The inhibitory effect of ethylamine was restored by increasing the concentration of potassium phosphate buffer in the mixture. Approximately 600 mM (110 mg/ml) theanine was formed in 48 h in an improved reaction mixture containing 600 mM sodium glutamate, 600 mM ethylamine.HCl, 300 mM glucose, 200 mM potassium phosphate buffer (pH 7.0), 30 mM MgCl(2), 5 mM MnCl(2), 5 mM AMP, 30 units/ml of GMAS, and 40 mg/ml of yeast cells. The yield of theanine was 100% on the substrates (glutamic acid and ethylamine) and also on the energy source (glucose consumed).     

Identification of yeasts present in sour fermented foods and fodders

This paper deals with rapid methods for identification of 50 yeast species frequently isolated from foods and fodders that underwent a lactic acid fermentation. However, many yeast species present in olive brine, alpechin, and other olive products were not treated. The methods required for identification include light microscopy, physiological growth tests (ID32C system of BioMérieux), assimilation of nitrate and of ethylamine as sole nitrogen sources, vitamin requirement, and maximum growth temperature. An identification key to treated yeast species is provided. In another table characteristics of all yeast species treated are listed.     

Identification of a new locus in the Escherichia coli cotransduction gap that represents a new genetic component of the L-asparagine utilization system

A temperature-sensitive Escherichia coli mutant defective for the ability to utilize L-asparagine as a sole nitrogen source was isolated after N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis. The mutation (asu) produces two distinct phenotypic effects. Mutant strains grow poorly at high temperature on minimal plates containing asparagine as the sole nitrogen source; this effect is greatly exacerbated by the presence of methionine. Mutant strains utilize L-asparagine as a nitrogen source three to four times more efficiently at permissive temperatures than the wild-type strains. The mutation maps at 32.4 min on the E. coli chromosome, within the E. coli cotransduction gap. Mutant strains produce normal amounts of thermo-stable L-asparaginase I activity. The mutation therefore affects a component of the asparagine utilization system other than the catabolism of asparagine within the cell; it probably affects asparagine uptake.     

Isolation and characterization of a bacterium which utilizes polyester polyurethane as a sole carbon and nitrogen source

Abstract Various soil samples were screened for the presence of microorganisms which have the ability to degrade polyurethane compounds. Two strains with good polyurethane degrading activity were isolated. The more active strain was tentatively identified as Comamonas acidovorans . This strain could utilize polyester-type polyurethanes but not the polyether-type polyurethanes as sole carbon and nitrogen sources. Adipic acid and diethylene glycol were probably the main degradation products when polyurethane was supplied as a sole carbon and nitrogen source. When ammonium nitrate was used as nitrogen source, only diethylene glycol was detected after growth on polyurethane.     

Evaluation of alternative nitrogen and carbon sources for sugarbeet suspension culture platings in development of cell selection schemes

Summary Low molecular weight nitrogenous impurity compounds as well as raffinose are negative quality factors that interfere with efficient processing of sugarbeet (Beta vulgaris L.) for sucrose. In order to identify nutrient media for cell selection of biochemical mutants or transgenics that might have reduced levels of these processing impurities, the ability of 10 endogenous compounds to serve as sole nitrogen or carbon source for suspension plating and subculture callus growth was evaluated. The most productive concentrations of nitrate, ammonium, l-glutamine, l-glutamate, urea, and l-proline as sole nitrogen sources supported plating callus growth at 106, 159, 233, 167, 80, and 52%, respectively, as well as the historical 60 mM mix of nitrate and ammonium in Murashige-Skoog medium. Glycine betaine and choline did not support growth. d(+) Raffinose and d(+) galactose supported plating callus growth only 67 and 25%, respectively, as well as sucrose as sole carbohydrate source. No callus growth occurred on glutamine, glutamate, or glycine betaine as the sole carbon or carbon plus nitrogen source. Platings on either nitrate or ammonium as sole nitrogen source did not differ in sensitivity to the nitrate uptake inhibitor phenylglyoxal, suggesting that phenylglyoxal lacks the specificity for use in selection for mutants of nitrate uptake. The ability of raffinose to be used as the carbon source, and glutamine or glutamate as the nitrogen source, may preclude their use for selection of genetic variants accumulating less of these processing impurities. However, mutants or transgenics able to utilize either glutamine, glutamate, or glycine betaine might be selectable on media containing any one of these as carbon, nitrogen, or carbon plus nitrogen source, respectively, that is incapable of supporting wild-type cell growth.     

Nitrogen Source Activates TOR (Target of Rapamycin) Complex 1 via Glutamine and Independently of Gtr/Rag Proteins

The evolutionary conserved TOR complex 1 (TORC1) activates cell growth in response to nutrients. In yeast, TORC1 responds to the nitrogen source via a poorly understood mechanism. Leucine, and perhaps other amino acids, activates TORC1 via the small GTPases Gtr1 and Gtr2, orthologs of the mammalian Rag GTPases. Here we investigate the activation of TORC1 by the nitrogen source and how this might be related to TORC1 activation by Gtr/Rag. The quality of the nitrogen source, as defined by its ability to promote growth and glutamine accumulation, directly correlates with its ability to activate TORC1 as measured by Sch9 phosphorylation. Preferred nitrogen sources stimulate rapid, sustained Sch9 phosphorylation and glutamine accumulation. Inhibition of glutamine synthesis reduces TORC1 activity and growth. Poor nitrogen sources stimulate rapid but transient Sch9 phosphorylation. A Gtr1 deficiency prevents the transient stimulation of TORC1 but does not affect the sustained TORC1 activity in response to good nitrogen sources. These findings suggest that the nitrogen source must be converted to glutamine, the preferred nitrogen source in yeast, to sustain TORC1 activity. Furthermore, sustained TORC1 activity is independent of Gtr/Rag. Thus, the nitrogen source and Gtr/Rag activate TORC1 via different mechanisms.     

A study of the properties of the free amino acid pool and enzyme synthesis in yeast

A study has been made of the distribution and properties of the free amino acid pool in yeast. The depletion of the pool was found to depend upon the energy source used, conditions of growth, and the nature of the exogenous nitrogen source. Pool levels could be restored either by an internal replenishment mechanism or by various nitrogen sources. In the absence of internal replenishment a strong positive correlation was established between the ability of nitrogen compounds to support free glutamic add synthesis and enzyme-synthesizing capacity. Amino acid assimilation by nitrogen-starved yeast was studied and compared with that in other organisms. The significance of these results for the problem of enzyme and protein synthesis in yeast is discussed.     

Carbon source utilization and inhibitor tolerance of 45 oleaginous yeast species

Conversion of lignocellulosic hydrolysates to lipids using oleaginous (high lipid) yeasts requires alignment of the hydrolysate composition with the characteristics of the yeast strain, including ability to utilize certain nutrients, ability to grow independently of costly nutrients such as vitamins, and ability to tolerate inhibitors. Some combination of these characteristics may be present in wild strains. In this study, 48 oleaginous yeast strains belonging to 45 species were tested for ability to utilize carbon sources associated with lignocellulosic hydrolysates, tolerate inhibitors, and grow in medium without supplemented vitamins. Some well-studied oleaginous yeast species, as well as some that have not been frequently utilized in research or industrial production, emerged as promising candidates for industrial use due to ability to utilize many carbon sources, including Cryptococcus aureus, Cryptococcus laurentii, Hannaella aff. zeae, Tremella encephala, and Trichosporon coremiiforme. Other species excelled in inhibitor tolerance, including Candida aff. tropicalis, Cyberlindnera jadinii, Metschnikowia pulcherrima, Schwanniomyces occidentalis and Wickerhamomyces ciferrii. No yeast tested could utilize all carbon sources and tolerate all inhibitors tested. These results indicate that yeast strains should be selected based on characteristics compatible with the composition of the targeted hydrolysate. Other factors to consider include the production of valuable co-products such as carotenoids, availability of genetic tools, biosafety level, and flocculation of the yeast strain. The data generated in this study will aid in aligning yeasts with compatible hydrolysates for conversion of carbohydrates to lipids to be used for biofuels and other oleochemicals.     

Degradation of o-toluidine by Rhodococcus rhodochrous

Abstract A Gram-positive bacterium with the ability to utilize o -toluidine as sole source of carbon and nitrogen was isolated from soil. The organism was identified as Rhodococcus rhodochrous Sb 4. 3-Methylcatechol and the meta-fission product of 3-methylcatechol were identified as metabolites. A pathway for the degradation of o -toluidine is proposed.     

Aerobic Degradation of 2,4,6-Trinitrotoluene by Enterobacter cloacae PB2 and by Pentaerythritol Tetranitrate Reductase

Enterobacter cloacae PB2 was originally isolated on the basis of its ability to utilize nitrate esters, such as pentaerythritol tetranitrate (PETN) and glycerol trinitrate, as the sole nitrogen source for growth. The enzyme responsible is an NADPH-dependent reductase designated PETN reductase. E. cloacae PB2 was found to be capable of slow aerobic growth with 2,4,6-trinitrotoluene (TNT) as the sole nitrogen source. Dinitrotoluenes were not produced and could not be used as nitrogen sources. Purified PETN reductase was found to reduce TNT to its hydride-Meisenheimer complex, which was further reduced to the dihydride-Meisenheimer complex. Purified PETN reductase and recombinant Escherichia coli expressing PETN reductase were able to liberate nitrogen as nitrite from TNT. The ability to remove nitrogen from TNT suggests that PB2 or recombinant organisms expressing PETN reductase may be useful for bioremediation of TNT-contaminated soil and water.