Antimicrobial activity of Monascus pilosus IFO 4520 against contaminant of Koji.

Antimicrobial activity of Monascus pilosus IFO 4520 was examined to prevent contamination during beni-koji making in the open air. The antibacterial effect of the beni-koji prepared with this strain occured with 30 mg/ml of beni-koji extract in combination with 0.5% lactic acid against two contaminants of koji, Micrococcus varians and Bacillus subtilis. There were two compounds, antibacterial and antiyeast substances, in the beni-koji extract. These results suggest a possibility of inhibiting the growths of contaminants during beni-koji making using beni-koji extract and lactic acid.     

Cerulenin-resistant mutants of Saccharomyces cerevisiae with an altered fatty acid synthase gene

Cerulenin, an antifungal antibiotic produced by Cephalosporium caerulens, is a potent inhibitor of fatty acid synthase in various organisms, including Saccharomyces cerevisiae. The antibiotic inhibits the enzyme by binding covalently to the active center cysteine of the condensing enzyme domain. We isolated 12 cerulenin-resistant mutants of S. cerevisiae following treatment with ethyl methanesulfonate. The mechanism of cerulenin resistance in one of the mutants, KNCR-1, was studied. Growth of the mutant was over 20 times more resistant to cerulenin than that of the wild-type strain. Tetrad analysis suggested that all mutants mapped at the same locus, FAS2, the gene encoding the α subunit of the fatty acid synthase. The isolated fatty acid synthase, purified from the mutant KNCR-1, was highly resistant to cerulenin. The cerulenin concentration causing 50% inhibition (IC₅₀) of the enzyme activity was measured to be 400 μM, whereas the IC₅₀ value was 15 μM for the enzyme isolated from the wild-type strain, indicating a 30-fold increase in resistance to cerulenin. The FAS2 gene was cloned from the mutant. Sequence replacement experiments suggested that an 0.8 kb EcoRV-HindIII fragment closely correlated with cerulenin resistance. Sequence analysis of this region revealed that the GGT codon encoding Gly-1257 of the FAS2 gene was altered to AGT in the mutant, resulting in the codon for Ser. Furthermore, a recombinant FAS2 gene, in which the 0.8 Kb EcoRV-HindIII fragment of the wild-type FAS2 gene was replaced with the same region from the mutant, when introduced into FAS2-defective S. cerevisiae complemented the FAS2 pheno-type and showed cerulenin resistance. These data indicate that one amino acid substitution (Gly → Ser) in the α subunit of fatty acid synthase is responsible for the cerulenin resistance of the mutant KNCR-1.     

Changes in gamma-aminobutyric acid content during beni-koji making

The changes in the gamma-aminobutyric acid (GABA) content during the making of beni-koji prepared with Monascus pilosus IFO 4520 vs. the difference in the rate of tomo koji (10%, 30%, and 50%) were examined. The increased proportion of tomo koji would increase the GABA production and the productions of GABA peaked on the fifth day and thereafter declined. The glutamate decarboxylase activity during beni-koji making with 50% tomo koji steadily increased after the start of the koji making, reaching its peak on the fifth day. The succinic acid content increased after the sixth day. The mycelial growth was in the stationary phase after the sixth day. Therefore, the GABA content increases with an increase in the proportion of tomo koji. It is presumed that the maximum amount of GABA reached on the fifth day was the cause of the increasing amount of conversion of GABA into succinic acid, in addition to the decline in the GAD activity after the fifth day of koji making.     

Cerulenin-resistant mutants of Saccharomyces cerevisiae with an altered fatty acid synthase gene

Cerulenin, an antifungal antibiotic produced by Cephalosporium caerulens, is a potent inhibitor of fatty acid synthase in various organisms, including Saccharomyces cerevisiae. The antibiotic inhibits the enzyme by binding covalently to the active center cysteine of the condensing enzyme domain. We isolated 12 cerulenin-resistant mutants of S. cerevisiae following treatment with ethyl methanesulfonate. The mechanism of cerulenin resistance in one of the mutants, KNCR-1, was studied. Growth of the mutant was over 20 times more resistant to cerulenin than that of the wild-type strain. Tetrad analysis suggested that all mutants mapped at the same locus, FAS2, the gene encoding the subunit of the fatty acid synthase. The isolated fatty acid synthase, purified from the mutant KNCR-1, was highly resistant to cerulenin. The cerulenin concentration causing 50% inhibition (IC₅₀) of the enzyme activity was measured to be 400 M, whereas the IC₅₀ value was 15 M for the enzyme isolated from the wild-type strain, indicating a 30-fold increase in resistance to cerulenin. The FAS2 gene was cloned from the mutant. Sequence replacement experiments suggested that an 0.8 kb EcoRV-HindIII fragment closely correlated with cerulenin resistance. Sequence analysis of this region revealed that the GGT codon encoding Gly-1257 of the FAS2 gene was altered to AGT in the mutant, resulting in the codon for Ser. Furthermore, a recombinant FAS2 gene, in which the 0.8 Kb EcoRV-HindIII fragment of the wild-type FAS2 gene was replaced with the same region from the mutant, when introduced into FAS2-defective S. cerevisiae complemented the FAS2 pheno-type and showed cerulenin resistance. These data indicate that one amino acid substitution (Gly Ser) in the subunit of fatty acid synthase is responsible for the cerulenin resistance of the mutant KNCR-1.     

Inhibition of Vibrio harveyi bioluminescence by cerulenin: in vivo evidence for covalent modification of the reductase enzyme involved in aldehyde synthesis.

Bacterial bioluminescence is very sensitive to cerulenin, a fungal antibiotic which is known to inhibit fatty acid synthesis. When Vibrio harveyi cells pretreated with cerulenin were incubated with [3H]myristic acid in vivo, acylation of the 57-kilodalton reductase subunit of the luminescence-specific fatty acid reductase complex was specifically inhibited. In contrast, in vitro acylation of both the synthetase and transferase subunits, as well as the activities of luciferase, transferase, and aldehyde dehydrogenase, were not adversely affected by cerulenin. Light emission of wild-type V. harveyi was 20-fold less sensitive to cerulenin at low concentrations (10 micrograms/ml) than that of the dark mutant strain M17, which requires exogenous myristic acid for luminescence because of a defective transferase subunit. The sensitivity of myristic acid-stimulated luminescence in the mutant strain M17 exceeded that of phospholipid synthesis from [14C]acetate, whereas uptake and incorporation of exogenous [14C]myristic acid into phospholipids was increased by cerulenin. The reductase subunit could be labeled by incubating M17 cells with [3H]tetrahydrocerulenin; this labeling was prevented by preincubation with either unlabeled cerulenin or myristic acid. Labeling of the reductase subunit with [3H]tetrahydrocerulenin was also noted in an aldehyde-stimulated mutant (A16) but not in wild-type cells or in another aldehyde-stimulated mutant (M42) in which [3H]myristoyl turnover at the reductase subunit was found to be defective. These results indicate that (i) cerulenin specifically and covalently inhibits the reductase component of aldehyde synthesis, (ii) this enzyme is partially protected from cerulenin inhibition in the wild-type strain in vivo, and (iii) two dark mutants which exhibit similar luminescence phenotypes (mutants A16 and M42) are blocked at different stages of fatty acid reduction.     

Self-cloning yeast strains containing novel FAS2 mutations produce a higher amount of ethyl caproate in Japanese sake

Point mutation of Gly1250Ser (1250S) of the yeast fatty acid synthase gene FAS2 confers cerulenin resistance. This mutation also results in a higher production of the apple-like flavor component ethyl caproate in Japanese sake. We mutated the 1250th codon by in vitro site-directed mutagenesis to encode Ala (1250A) or Cys (1250C) and examined cerulenin resistance and ethyl caproate production. The mutated FAS2 genes were inserted into a binary plasmid vector containing a drug-resistance marker and a counter-selectable marker, GALp-GIN11M86. The plasmids were integrated into the wild-type FAS2 locus of a sake yeast strain, and the loss of the plasmid sequences from the integrants was done by growth on galactose plates, which is permissive for loss of GALp-GIN11M86. These counter-selected strains contained either the wild type or the mutated FAS2 allele but not the plasmid sequences, from which FAS2 mutant strains were selected by allele-specific PCR. The FAS2-1250C mutant produced a higher amount of ethyl caproate in sake than FAS2-1250S, while FAS2-1250A produced an ethyl caproate level intermediate between FAS2-1250S and the parental Kyokai no. 7 strain. Interestingly, these mutants only showed detectable cerulenin resistance. These 'self-cloning' yeast strains should be acceptable to the public because they can improve sake quality without the presence of extraneous DNA sequences.     

Effects of the principal nutrients on lovastatin production by Monascus pilosus

Lovastatin production is dependent on the substrates provided. We investigated how several carbon and nitrogen sources in the medium affect lovastatin production by Monascus pilosus. M. pilosus required a suitable concentration of organic nitrogen peptone for high lovastatin production. As sole carbon source with peptone, although glucose strongly repressed lovastatin production, maltose was responsible for high production. Interestingly, glycerol combined with maltose enhanced lovastatin production, up to 444 mg/l in the most effective case. Moreover, an isolated mutant, in which glucose repression might be relieved, easily produced the highest level of lovastatin, 725 mg/l on glucose-glycerol-peptone medium. These observations indicate that lovastatin production by M. pilosus is regulated by strict glucose repression and that an appropriate release from this repression by optimizing medium composition and/or by a mutation(s) is required for high lovastatin production.     

Molecular cloning of the cDNA for a mutant mouse ribonucleotide reductase M1 that produces a dominant mutator phenotype in mammalian cells.

Mammalian ribonucleotide reductase is regulated by the binding of dATP and other nucleotide effectors to allosteric sites on subunit M1. Using mRNA from a mutant mouse T-lymphoma (S49) cell line, we have isolated a cDNA which encodes an altered, dATP feedback-resistant subunit M1. The mutant cDNA contains a single point mutation (a G-to-A transition) at codon 57, converting aspartic acid to asparagine. Proof that this mutation is responsible for the phenotype of dATP feedback resistance is provided by the following evidence. (i) The mutation was detected only in mutant S49 cells containing dATP feedback-resistant ribonucleotide reductase and not in wild-type or other mutant S49 cells. (ii) Transfection of Chinese hamster ovary cells with an expression plasmid containing the mutant M1 cDNA resulted in the production of dATP feedback-resistant ribonucleotide reductase. Transfected CHO cells expressing the mutant M1 cDNA exhibited a 15- to 25-fold increase in the frequency of spontaneous mutation to 6-thioguanine resistance, confirming that dATP feedback-resistant ribonucleotide reductase produces a mutator phenotype in mammalian cells. The availability of a cDNA which encodes dATP feedback-resistant subunit M1 thus provides a means of manipulating by transfection the frequency of spontaneous mutation in mammalian cells.     

Thiolactomycin resistance in Escherichia coli is associated with the multidrug resistance efflux pump encoded by emrAB.

Thiolactomycin (TLM) and cerulenin are antibiotics that block Escherichia coli growth by inhibiting fatty acid biosynthesis at the beta-ketoacyl-acyl carrier protein synthase I step. Both TLM and cerulenin trigger the accumulation of intracellular malonyl-coenzyme A coincident with growth inhibition, and the overexpression of synthase I protein confers resistance to both antibiotics. Strain CDM5 was derived as a TLM-resistant mutant but remained sensitive to cerulenin. TLM neither induced malonyl-coenzyme A accumulation nor blocked fatty acid production in vivo; however, the fatty acid synthase activity in extracts from strain CDM5 was sensitive to TLM inhibition. The TLM resistance gene in strain CDM5 was mapped to 57.5 min of the chromosome and was an allele of the emrB gene. Disruption of the emrB gene converted strain CDM5 to a TLM-sensitive strain, and the overexpression of the emrAB operon conferred TLM resistance to sensitive strains. Thus, activation of the emr efflux pump is the mechanism for TLM resistance in strain CDM5.     

Transfer of 'Thermoactinomyces glaucus' IFO 12530 and 'Thermoactinomyces monosporus' IFO 14050 to the genus Saccharomonospora as members of Saccharomonospora glauca

Two available strains of 'Thermoactinomyces glaucus' and 'Thermoactinomyces monosporus', 'T. glaucus' IFO 12530 and 'T. monosporus' IFO 14050, were considered not to be members of the genus Thermoactinomyces and that they belonged to the genus Saccharomonospora on the basis of the colors of colonies and 16S rDNA sequences. Some chemotaxonomic characteristics also showed that the two strains belong to the genus Saccharomonospora. The two strains contained meso-diaminopimelic acid, galactose, and arabinose in the cell wall and MK-9(H(4)) as the predominant menaquinone. The genomic DNAs of the two strains had a G+C content of 69 mol%. The 16S rDNAs of 'T. glaucus' IFO 12530 and 'T. monosporus' IFO 14050 showed only 1 and 2 bp sequence differences, respectively, from that of the type strain of Saccharomonospora glauca. Furthermore, the two strains of 'T. glaucus' and 'T. monosporus' and the type strain of S. glauca shared identical 16S-23S rDNA ITS sequences. The levels of DNA-DNA relatedness confirm that the two strains of 'T. glaucus' and 'T. monosporus' are members of Saccharomonospora glauca. Therefore it is proposed that 'T. glaucus' IFO 12530 and 'T. monosporus' IFO 14050 should be considered as strains belonging to Saccharomonospora glauca.     

Enhancement of polyunsaturated fatty acid production by cerulenin treatment in polyunsaturated fatty acid-producing bacteria

When docosahexaenoic acid (DHA)-producing Moritella marina strain MP-1 was cultured in the medium containing 0.5 μ g cerulenin ml⁻¹, an inhibitor for fatty acid biosynthesis, the cells grew normally, but the␣content of DHA in the total fatty acids increased from 5.9–19.4%. The DHA yield of M. marina strain MP-1 cells also increased from 4 to 13.7 mg l⁻¹ by cerulenin treatment. The same effect of cerulenin was observed in eicosapentaenoic acid (EPA)-producing Shewanella marinintestina strain IK-1 grown in the medium containing 7.5 μg cerulenin ml⁻¹, and the cerulenin treatment increased the EPA yield from 1.6 to 8 mg l⁻¹. The use of cerulenin is, therefore, advantageous to increase the content of intracellular polyunsaturated fatty acids (PUFA) in particular PUFA-containing phospholipids in bacterial cells.An erratum to this article can be found at .     

Selection of an effective red-pigment producing Monascus pilosus by efficient transformation with aurintricarboxylic acid

The filamentous fungus Monascus pilosus was genetically transformed with a reporter plasmid, pMS-1.5hp, by aurintricarboxylic acid (ATA) treatment to obtain an efficient red-pigment producing mutant. The transformation efficiency of Monascus pilosus was higher with the ATA-treatment than with either a non-restriction-enzyme-mediated integration (REMI) or a REMI method. This valid and convenient random mutagenesis method shows that ATA can be applied in fungi for efficient genetic transformation.     

Next-generation Akt inhibitors provide greater specificity: effects on glucose metabolism in adipocytes

Many human tumours exhibit activation of the PI3K (phosphoinositide 3-kinase)/Akt pathway, and inhibition of this pathway slows tumour growth. This led to the development of specific Akt inhibitors for in vivo use. However, activation of Akt is also necessary for processes including glucose metabolism. Therefore a potential complication of such anticancer drugs is insulin resistance and/or diabetes. In the process of characterizing the metabolic effects of early-phase Akt inhibitors, we discovered an off-target inhibitory effect on mammalian facilitative glucose transporters. In view of the crucial role of glucose transport for all mammalian cells, such an off-target effect would have major implications for further development of this family of compounds. In the present study, we have characterized a next-generation Akt inhibitor, MK-2206. MK-2206 is an orally active allosteric Akt inhibitor under development for treating solid tumours. We report that MK-2206 potently inhibits Thr308Akt and Ser473Akt phosphorylation in 3T3-L1 adipocytes (IC50 0.11 and 0.18 μM respectively) as well as downstream effects of insulin on GLUT4 (glucose transporter 4) translocation (IC50 0.47 μM) and glucose transport (IC50 0.14 μM). Notably, the potency of MK-2206 is approximately 1 log higher than previous inhibitors and its specificity is significantly improved with modest inhibitory effects on glucose transport in GLUT4-expressing adipocytes and GLUT1-rich human erythrocytes, independently of Akt. Nevertheless, MK-2206 clearly has potent effects on Akt2, the principal isoform involved in peripheral insulin action, in which case insulin resistance will probably be a major complication following in vivo administration. We conclude that MK-2206 provides an optimal tool for studying the effects of Akt in vitro.     

Demonstration of the physiological role of autolysis by a comparative study with a wild-type and its non-autolytic mutant of Micrococcus lysodeikticus (luteus) cultivated with externally added proteolytic enzymes

The log phase cells of autolytic Microccus lysodeikticus (luteus) IFO 3333 did not autolyze when grown in the presence of trypsin although the growth curve and morphology of the cells were not influenced. A non-autolytic mutant was obtained by subculture of the wild-type strain IFO 3333 on an agar slant containing 1% glucose. The mutant (strain MT) was wild-type IFO 3333 which occurred singly or in irregular masses. The mutant MT grown in a culture medium containing trypsin caused remarkable alteration in cell morphology: large cell packets consisting of a number of "unit tetrads" arranged regularly in three dimensions were formed by the addition of trypsin to the medium. The findings suggest that inhibition of the separation of divided cells is brought about by inactivation or suppression of a cell wall autolytic enzyme which plays an important role in the separation step and is accessible to externally added trypsin in the mutant cells but not in the wild-type cells. The possibility that there are two kinds or phases of autolytic enzymes "a physiological autolytic enzyme" and "a useless autolytic enzyme", is discussed.     

Cell separation system studied by mixed culture of single wild strain with tetrads-forming mutant strain of Micrococcus luteus

Mixed culture study of singly occurring wild strain IFO 3333 of Micrococcus luteus and a tetrads-forming mutant strain MT, in the absence or presence of trypsin, supported our previous assumption that at least two kinds of separation systems were involved in cell separation of M. luteus, the one having a physiological role in cutting off the outermost layer of the cell wall (separation system-Om) and the other in cutting off the inner layer of the "proper" cell wall or the septum (separation system-In). The separation system-Om of IFO 3333 insensitive to trypsin substituted, freely from the cells, for that of MT sensitive to trypsin.     

Inhibition of unsaturated fatty acid synthesis in escherichia coli by the antibiotic cerulenin.

Low concentrations of cerulenin inhibit the growth of Escherichia coli by selectively blocking unsaturated fatty acid synthesis. This inhibition was relieved by unsaturated fatty acid supplements alone but not by saturated fatty acid supplements. The utilization of exogenous unsaturated fatty acids to sustain growth in the presence of cerulenin was confirmed by the analysis of bulk lipid composition. The effects of cerulenin on fatty acid synthesis were examined in vivo by pulse labeling with [14C]acetate and in vitro using [14C]malonyl-coenzyme A. In both cases, unsaturated fatty acid synthesis was inhibited by low concentrations of cerulenin with a stimulation of saturated fatty acid synthesis. Using mutant strains deficient in fatty acid synthesis, the effects of cerulenin on beta-ketoacyl-[acyl-carrier-protein] synthetases I and II were examined. Our results indicate that beta-ketoacyl-[acyl-carrier-protein] synthetase I is more sensitive to inhibition by cerulenin than beta-ketoacyl-[acyl-carrier-protein] synthetase II.     

VSV disrupts the Rae1/mrnp41 mRNA nuclear export pathway

Interference with nucleocytoplasmic transport is a strategy employed by certain viruses to compromise host cellular function. While it has been shown that the matrix (M) protein of the vesicular stomatitis virus (VSV) inhibits nuclear export of host cell mRNAs, the underlying mechanism has not been fully established. Here we show that VSV M protein binds the mRNA export factor Rae1/mrnp41. A mutant of M protein defective in Rae1 binding is unable to inhibit mRNA nuclear export. We further show that increased expression of Rae1 fully reverts the inhibition of mRNA export induced by M protein or following virus infection. We found that Rae1 is induced by interferon-gamma, a cytokine that plays a critical role in the immune response to viruses, such as VSV. Thus, these results demonstrate that VSV M protein blocks mRNA export by disrupting Rae1 function, which can be reverted by induction of Rae1 expression.