Micellar Mechanisms for Desymmetrization Reactions in Aqueous Media.

Water-mediated organic reactions significantly contribute to the protection of the environment. Desymmetrization reactions, which convert only one of the identical functional groups within one molecule, are cost-effective because of the low cost of the starting materials. In combination with these two merits, highly efficient and practical selective monohydrolysis reactions of symmetric diesters were previously reported. The products of these reactions are versatile building blocks. The mechanisms of this reaction are hypothesized to proceed through micellar aggregates in which the hydrophilic carboxylate anions formed by monohydrolysis are directed outward and the remaining hydrophobic groups are directed inward in governing the selectivities. Here, dynamic light scattering and electrophoretic light scattering experiments were performed for detection of the key intermediates in the reaction. These experiments revealed the existence of aggregates with negative charges on the surface in the mainly aqueous media, supporting the reaction mechanisms that control the high selectivities.

Neural dysfunctions following experimental permanent occlusions of bilateral common carotid arteries cause an increase of rat voluntary alcohol drinking behavior.

We have previously reported that ischemic animal models treated with a respiratory inhibitor, rotenon, show an increased voluntary alcohol intake. Although it is clear that ischemic brain, as a result of reduced-blood flow, shows pathological events and/or neuro-degenerations apparently, little is known of causal relationship between the mechanism of neural dysfunction and voluntary alcohol consumption. Authors have investigated effects of permanent two-vessel occlusion (p2VO) on rat voluntary alcohol drinking behavior. In first experiment the p2VO-treated rats showed an increase of voluntary alcohol drinking behavior, as compared with sham controls. Using brain microdialysis technique, increases of only nucleus accumbens (ACC) dopamine (DA) releases were suppressed in the p2VO-treated rats significantly, following the high K+ (40 mM) perfusion through the microdialysis probe membrane. Alcohol (200 mM) perfusion-induced DA and serotonin (5-HT) releases in the ACC of the p2VO-treated rats were suppressed significantly in the second experiment, as compared with the sham-treated rats. In third experiment p2VO-treated rats showed significant decreases of the contents of DA, not 5-HT, in the ACC, caudate-putamen (C/P), ventral tegmental area-substantia nigra (VT/SN) and lateral hypothalamus (LH). Dopaminergic neurons in the ACC showed more functional vulnerability against the p2VO treatments, as compared with the serotonergic neurons. An increase of alcohol intake in the p2VO-treated rats means the compensation for the neural degeneration of the dopaminergic system in the ACC consisted brain rewarding system. It was likely suggested that neural disturbance of higher functions involved with incomplete global brain ischemia leads the risk of an abnormal alcohol drinking in human.

Combined Drug Resistance Mutations Substantially Enhance Enzyme Production in Paenibacillus agaridevorans.

This study shows that sequential introduction of drug resistance mutations substantially increased enzyme production in Paenibacillus agaridevorans The triple mutant YT478 (rsmG Gln225→stop codon, rpsL K56R, and rpoB R485H), generated by screening for resistance to streptomycin and rifampin, expressed a 1,100-fold-larger amount of the extracellular enzyme cycloisomaltooligosaccharide glucanotransferase (CITase) than the wild-type strain. These mutants were characterized by higher intracellular S-adenosylmethionine concentrations during exponential phase and enhanced protein synthesis activity during stationary phase. Surprisingly, the maximal expression of CITase mRNA was similar in the wild-type and triple mutant strains, but the mutant showed greater CITase mRNA expression throughout the growth curve, resulting in enzyme overproduction. A metabolome analysis showed that the triple mutant YT478 had higher levels of nucleic acids and glycolysis metabolites than the wild type, indicating that YT478 mutant cells were activated. The production of CITase by the triple mutant was further enhanced by introducing a mutation conferring resistance to the rare earth element, scandium. This combined drug resistance mutation method also effectively enhanced the production of amylases, proteases, and agarases by P. agaridevorans and Streptomyces coelicolor This method also activated the silent or weak expression of the P. agaridevorans CITase gene, as shown by comparisons of the CITase gene loci of P. agaridevorans T-3040 and another cycloisomaltooligosaccharide-producing bacterium, Paenibacillus sp. strain 598K. The simplicity and wide applicability of this method should facilitate not only industrial enzyme production but also the identification of dormant enzymes by activating the expression of silent or weakly expressed genes.IMPORTANCE Enzyme use has become more widespread in industry. This study evaluated the molecular basis and effectiveness of ribosome engineering in markedly enhancing enzyme production (>1,000-fold). This method, due to its simplicity, wide applicability, and scalability for large-scale production, should facilitate not only industrial enzyme production but also the identification of novel enzymes, because microorganisms contain many silent or weakly expressed genes which encode novel antibiotics or enzymes. Furthermore, this study provides a new mechanism for strain improvement, with a consistent rather than transient high expression of the key gene(s) involved in enzyme production.

Metabolic perturbation to enhance polyketide and nonribosomal peptide antibiotic production using triclosan and ribosome-targeting drugs.

Although transcriptional activation of pathwayspecific positive regulatory genes and/or biosynthetic genes is primarily important for enhancing secondary metabolite production, reinforcement of substrate supply, as represented by primary metabolites, is also effective. For example, partial inhibition of fatty acid synthesis with ARC2 (an analog of triclosan) was found to enhance polyketide antibiotic production. Here, we demonstrate that this approach is effective even for industrial high-producing strains, for example enhancing salinomycin production by 40%, reaching 30.4 g/l of salinomycin in an industrial Streptomyces albus strain. We also hypothesized that a similar approach would be applicable to another important antibiotic group, nonribosomal peptide (NRP) antibiotics. We therefore attempted to partially inhibit protein synthesis by using ribosome-targeting drugs at subinhibitory concentrations (1/50∼1/2 of MICs), which may result in the preferential recruitment of intracellular amino acids to the biosynthesis of NRP antibiotics rather than to protein synthesis. Among the ribosome-targeting drugs examined, chloramphenicol at subinhibitory concentrations was most effective at enhancing the production by Streptomyces of NRP antibiotics such as actinomycin, calcium-dependent antibiotic (CDA), and piperidamycin, often resulting in an almost 2-fold increase in antibiotic production. Chloramphenicol activated biosynthetic genes at the transcriptional level and increased amino acid pool sizes 1.5- to 6-fold, enhancing the production of actinomycin and CDA. This "metabolic perturbation" approach using subinhibitory concentrations of ribosome-targeting drugs is a rational method of enhancing NRP antibiotic production, being especially effective in transcriptionally activated (e.g., rpoB mutant) strains. Because this approach does not require prior genetic information, it may be widely applicable for enhancing bacterial production of NRP antibiotics and bioactive peptides.

The mthA mutation conferring low-level resistance to streptomycin enhances antibiotic production in Bacillus subtilis by increasing the S-adenosylmethionine pool size.

Certain Str(r) mutations that confer low-level streptomycin resistance result in the overproduction of antibiotics by Bacillus subtilis. Using comparative genome-sequencing analysis, we successfully identified this novel mutation in B. subtilis as being located in the mthA gene, which encodes S-adenosylhomocysteine/methylthioadenosine nucleosidase, an enzyme involved in the S-adenosylmethionine (SAM)-recycling pathways. Transformation experiments showed that this mthA mutation was responsible for the acquisition of low-level streptomycin resistance and overproduction of bacilysin. The mthA mutant had an elevated level of intracellular SAM, apparently acquired by arresting SAM-recycling pathways. This increase in the SAM level was directly responsible for bacilysin overproduction, as confirmed by forced expression of the metK gene encoding SAM synthetase. The mthA mutation fully exerted its effect on antibiotic overproduction in the genetic background of rel(+) but not the rel mutant, as demonstrated using an mthA relA double mutant. Strikingly, the mthA mutation activated, at the transcription level, even the dormant ability to produce another antibiotic, neotrehalosadiamine, at concentrations of 150 to 200 µg/ml, an antibiotic not produced (<1 µg/ml) by the wild-type strain. These findings establish the significance of SAM in initiating bacterial secondary metabolism. They also suggest a feasible methodology to enhance or activate antibiotic production, by introducing either the rsmG mutation to Streptomyces or the mthA mutation to eubacteria, since many eubacteria have mthA homologues.

Hydroxy-group effect on the regioselectivity in a photochemical oxetane formation reaction (the Paternò-Büchi Reaction) of geraniol derivatives.

The Paternò-Büchi (PB) reaction of geraniol derivatives 1, which contain allylic alcohol functionality and unfunctionalized double bonds, with benzophenone was investigated to see the effect of the hydroxyl group on the regioselectivity of the oxetane formation, i.e., 2/3. At low concentration of geraniol (1a), oxetanes 2a and 3a were formed in a ratio of 2a/3a = ca. 50/50. The oxetane 2a is derived from the PB reaction at the allylic alcohol moiety, whereas the PB reaction at the unfunctionalized double bond produces the oxetane 3a. The PB reaction of the hydroxy-protected methyl ether 1b and acetate 1c gave selectively oxetanes 3b,c derived from the reaction at the more nucleophilic double bond, 2/3∼ 15/85. The hydroxyl-group effect was found to be small, but apparently increased the formation of 2a in the PB reaction with geraniol (1a).

Formation of macrocyclic lactones in the Paternò-Büchi dimerization reaction.

Furan-2-ylmethyl 2-oxoacetates 1a,b, in which the furan ring and the carbonyl moiety were embedded intramolecularly, were synthesized from commercially available furan-2-ylmethanol and their photochemical reaction (hν > 290 nm) was investigated. Twelve-membered macrocyclic lactones 2a,b with C(i) symmetry including two oxetane-rings, which are the Paternò-Büchi dimerization products, were isolated in ca. 20% yield. The intramolecular cyclization products, such as 3-alkoxyoxetane and 2,7-dioxabicyclo[2.2.1]hept-5-ene derivatives, were not detected in the photolysate.

Concentration and temperature dependency of regio- and stereoselectivity in a photochemical [2 + 2] cycloaddition reaction (the Paternò-Büchi reaction): origin of the hydroxy-group directivity.

A set of photochemical oxetane formation reactions, i.e., the Paternò-Büchi (PB) reactions, of tetrahydrobenzofuranol derivatives 1a-d with benzophenone (BP) was investigated to examine poorly understood hydroxy-group directivity on regio- and stereoselectivity. The selectivities of the PB reactions for allylic alcohols 1a,d were found to be largely dependent upon the concentration of the allylic alcohols and the reaction temperature. The temperature-dependent change of the regioselectivity at high concentrations of allylic alcohols was similar to that of the hydroxy-protected methyl ether 1b and tetrahydrobenzofuran (1c). The effect of concentration on regioselectivity can be explained by the hydrogen-bonded aggregates, which mimic the selectivities observed during the PB reaction of 1b,c. The hydroxy-directed cis-selectivity of the higher-substituted oxetane 3a,d formed at low concentration of 1a,d was found to be larger than that at the higher concentration of 1a,d. The cis-selectivity of 3a,d was found to be higher than that of the lower-substituted oxetane 2a,d. The regioselectivity of the cis-configured oxetanes was higher than that of the trans-configured oxetanes. These experimental results strongly suggest that hydroxy-group directivity, induced by hydrogen-bonding stabilization, plays a role in controlling the regio- and stereoselectivity of the PB reactions. The steric effect was also responsible for the diastereoselectivity, as shown by the fact that the cis selectivity in 3d was higher than that in 3a. Computational studies at the (U)MP2 and (U)DFT level of theory revealed that hydrogen-bonding stabilization becomes important only in the excited complex (exciplex) between the triplet excited state of carbonyls and alkenes, in which the charge transfer occurs from the alkene to the excited carbonyl to make the carbonyl oxygen nucleophilic. No significant stabilization energy was found in the intermediary triplet state of biradicals. The combined experimental and computational studies have clarified the origin of the poorly understood hydroxy-group effect on a high degree of regio- and stereoselectivity, i.e., the cooperative effect of hydrogen-bonding stabilization in exciplexes and the steric bulk of the substituents.

Contribution of 1,2-dihydroxy-5-(methylsulfinyl)pentan-3-one (DMTS-P1) to the formation of dimethyl trisulfide (DMTS) during the storage of Japanese sake.

Dimethyl trisulfide (DMTS) is involved in the unpalatable aroma of stale Japanese sake, called "hineka". Recently, we isolated one of the precursor compounds of DMTS in sake and identified it as 1,2-dihydroxy-5-(methylsulfinyl)pentan-3-one (DMTS-P1), a previously unknown compound. In this work, the contribution of DMTS-P1 to the formation of DMTS was investigated. DMTS-P1 was chemically synthesized from methional in three steps, consisting of the Grignard reaction, followed by oxidation by MnO(2) and an immobilized osmium oxide catalyst. The formation of synthetic DMTS-P1 was confirmed by a comparison of the liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR) data to that of natural DMTS-P1. Quantitative analysis of DMTS-P1 in sake was developed using LC-MS/MS, and a positive correlation was observed between the concentration of DMTS-P1 in sake and the production of DMTS during storage. These results indicate that DMTS-P1 contributes to the formation of DMTS in sake.

Screening and identification of precursor compounds of dimethyl trisulfide (DMTS) in Japanese sake.

Dimethyl trisulfide (DMTS) is involved in the unpalatable aroma of stale sake, called "hineka"; however, the mechanism underlying the formation of DMTS during the storage of sake has not been elucidated. This paper investigates the precursors of DMTS in sake. An experiment using [methyl-d(3)]-methionine showed that Strecker degradation of methionine plays a minor role in the formation of DMTS. Separation of components in sake by cation exchange resin revealed that DMTS precursors are present in the acidic/neutral fraction rather than in the basic one. Purification of the DMTS precursor compounds was carried out through several chromatographic steps, measuring DMTS-producing potential as an index. High-resolution ESI-MS and 1D/2D NMR experiments enabled the identification of one of the precursor compounds as 1,2-dihydroxy-5-(methylsulfinyl)pentan-3-one.

Three new glycolipids with cytolytic activity from cultured marine dinoflagellate Heterocapsa circularisquama.

A monogalactosyl monoacylglycerol 1 and two digalactosyl monoacylglycerols 2 and 3 were isolated from a cultured marine dinoflagellate Heterocapsa circularisquama along with known (2S)-1-O-6,9,12,15-octadecatetraenoyl-3-O-beta-D-galactopyranosyl]-sn-glycerol (4). On the basis of spectral analysis, the glycolipid 1 was characterised as (2S)-1-O-3,6,9,12,15-octadecapentaenoyl-3-O-beta-D-galactopyranosyl]-sn-glycerol. The glycolipids 2 and 3 were characterised as (2S)-1-O-3,6,9,12,15-octadecapentaenoyl-3-O-[alpha-D-galactopyranosyl-(1''' --> 6'')-O-beta-D-galactopyranosyl]-sn-glycerol and (2S)-1-O-6,9,12,15-octadecatetraenoyl-3-O-[alpha-D-galactopyranosyl-(1''' --> 6'')-O-beta-D-galactopyranosyl]-sn-glycerol, respectively. The isolated monoacylglycerols 1-4 showed cytolytic activity towards heart and gill cells of oyster.

Multiple functions of ergosterol in the fission yeast Schizosaccharomyces pombe.

Sterols are a major class of membrane lipids in eukaryotes. In Schizosaccharomyces pombe, sterol 24-C-methyltransferase (Erg6p), C-8 sterol isomerase (Erg2p), C-5 sterol desaturase (Erg31p, Erg32p), C-22 sterol desaturase (Erg5p) and C-24 (28) sterol reductase (Sts1p/Erg4p) have been predicted, but not yet determined, to catalyse a sequence of reactions from zymosterol to ergosterol. Disruption mutants of these genes were unable to synthesize ergosterol, and most were tolerant to the polyene drugs amphotericin B and nystatin. Disruption of erg31(+) or erg32(+) did not cause ergosterol deficiency or tolerance to polyene drugs, indicating that the two C-5 sterol desaturases have overlapping functions. GFP-tagged DRM (detergent-resistant membrane)-associated protein Pma1p localized to the plasma membrane in ergDelta mutants. DRM fractionation revealed that the association between Pma1-GFP and DRM was weakened in erg6Delta but not in other erg mutants. Several GFP-tagged plasma membrane proteins were tested, and an amino acid permease homologue, SPBC359.03c, was found to mislocalize to intracellular punctate structures in the ergDelta mutants. These results indicate that these proteins are responsible for ergosterol biosynthesis in fission yeast, similar to the situation in Saccharomyces cerevisiae. Furthermore, in fission yeast, ergosterol is important for plasma membrane structure and function and for localization of plasma membrane proteins.

A new method for isolation of S-adenosylmethionine (SAM)-accumulating yeast.

S-Adenosylmethionine (SAM) is an important metabolite that participates in many reactions as a methyl group donor in all organisms, and has attracted much interest in clinical research because of its potential to improve many diseases, such as depression, liver disease, and osteoarthritis. Because of these potential applications, a more efficient means is needed to produce SAM. Accordingly, we developed a positive selection method to isolate SAM-accumulating yeast in this study. In Saccharomyces cerevisiae, one of the main reactions consuming SAM is thought to be the methylation reaction in the biosynthesis of ergosterol that is catalyzed by Erg6p. Mutants with deficiencies in ergosterol biosynthesis may accumulate SAM as a result of the reduction of SAM consumption in ergosterol biosynthesis. We have applied this method to isolate SAM-accumulating yeasts with nystatin, which has been used to select mutants with deficiencies in ergosterol biosynthesis. SAM-accumulating mutants from S. cerevisiae K-9 and X2180-1A were efficiently isolated through this method. These mutants accumulated 1.7-5.5 times more SAM than their parental strains. NMR and GC-MS analyses suggested that two mutants from K-9 have a mutation in the erg4 gene, and erg4 disruptants from laboratory strains also accumulated more SAM than their parental strains. These results indicate that mutants having mutations in the genes for enzymes that act downstream of Erg6p in ergosterol biosynthesis are effective in accumulating SAM.

Sterol composition of a cultured marine dinoflagellate, Heterocapsa circularisquama.

The composition of sterols was determined in a cultured marine dinoflagellate Heterocapsa circularisquama. Ten kinds of the sterol in H. circularisquama were identified by capillary gas chromatography-mass spectrometry. The major sterol was a 4-methyl sterol, 4alpha,23,24-trimethyl-5alpha-cholest-22E-en-3beta-ol (dinosterol) which is the common sterol in many dinoflagellates.

A new digalactosyl diacylglycerol from a cultured marine dinoflagellate Heterocapsa circularisquama.

A new digalactosyl diacylglycerol (1) has been isolated from the cultured marine dinoflagellate Heterocapsa circularisquama together with known monogalactosyl diacylglycerols 2 and 3, and their structures were elucidated by spectroscopic methods. The digalactosyl diacylglycerol 1 showed cytolytic activity toward heart cells of oysters.