Interaction of Organogermanium Compounds with Saccharides in Aqueous Solutions: Promotion of Aldose-to-ketose Isomerization and Its Molecular Mechanism.

This review discusses sugar isomerization with organogermanium compounds. Organogermanium compounds markedly increase the aldose-ketose (glucose-fructose or lactose-lactulose) isomerization ratio, double the initial reaction rate, and significantly reduce the base-catalyzed degradation of sugars. 1H-nuclear magnetic resonance analysis reveals that the affinity of organogermanium compounds with a 3-(trihydroxygermyl)propanoic acid (THGP) structure toward ketoses is 20-40 times stronger than that toward aldoses; thus, such organogermanium compounds form complexes more readily with ketoses than with aldoses. Stable ketose complexes, which contain multiple cis-diol structures and high fractions of furanose structures, suppress the reverse ketose-aldose reaction, thereby shifting the equilibrium toward the ketose side. These complexes also protect sugar molecules from alkaline degradation owing to the repulsion between anionic charges. The increased rate of the initial reaction in the alkaline isomerization process results from stabilizing the transition state by forming a complex between THGP and a cis-enediol intermediate. The cyclic pentacoordinate or hexacoordinate THGP structures give rise to a conjugated system of germanium orbitals, which is extended through dπ-pπ interactions, thereby improving the stability of the complex. Based on these results, we have developed a bench-scale lactulose syrup manufacturing plant incorporating a system to separate, recover, and reuse organogermanium poly-trans-[(2-carboxyethyl)germasesquioxane]. This manufacturing plant can be used as a model of an alkaline isomerization accelerator for continuous industrial production.

An Organogermanium Compound Enhances the Initial Reaction Rate of Alkaline Isomerization of an Aldose into a Ketose through Enediol Complex Formation.

We previously demonstrated that the organogermanium compound 3-(trihydroxygermyl)propanoic acid (THGP) enhances the enzymatic and alkaline isomerization of an aldose to a ketose through cis-diol complex formation by multiple mechanisms. Its higher affinity for the ketose than the aldose protects the ketose complex from alkaline decomposition. Furthermore, it has been reported that the aldose-ketose alkaline isomerization pathway includes 1,2-enediol. Therefore, we speculated that the complex-forming ability of THGP could also be applied to enediol, a transient intermediate of alkaline isomerization. To test this prediction, we analyzed the initial rates of glucose or lactose isomerization in a region where there was no substantial difference in pH with and without THGP addition. The results showed that THGP enhanced the rate of fructose or lactulose formation per unit time by approximately 2-fold compared to the control. This finding indicated that THGP could form a complex with the transition state of aldose-ketose alkaline isomerization.

Erythritol: Another C4 Platform Chemical in Biomass Refinery.

The potential of erythritol as a platform chemical in biomass refinery is discussed in terms of erythritol production and utilization. Regarding erythritol production, fermentation of sugar or starch has been already commercialized. The shift of the carbon source from glucose to inexpensive inedible waste glycerol is being investigated, which will decrease the price of erythritol. The carbon-based yield of erythritol from glycerol is comparable to or even higher than that from glucose. The metabolic pathway of erythritol biosynthesis has become clarified: erythrose-4-phosphate, which is one of the intermediates in the pentose phosphate pathway, is dephosphorylated and reduced to erythritol. The information about the metabolic pathway may give insights to improve the productivity by bleeding. Regarding erythritol utilization, chemical conversions of erythritol, especially deoxygenation, have been investigated in these days. Erythritol is easily dehydrated to 1,4-anhydroerythritol, which can be also used as the substrate for production of useful C4 chemicals. C-O hydrogenolysis and deoxydehydration using heterogeneous catalysts are effective reactions for erythritol/1,4-anhydroerythritol conversion.

Aspergillus luchuensis fatty acid oxygenase ppoC is necessary for 1-octen-3-ol biosynthesis in rice koji.

Awamori is a distilled spirit produced in Okinawa Prefecture, in southern Japan. Awamori contains the volatile organic compound 1-octen-3-ol, an important flavor component. Here, using solid-phase microextraction-gas chromatography-mass spectrometry (SPME-GCMS), we demonstrate that the black koji mold Aspergillus luchuensis produces 1-octen-3-ol in rice koji. To examine the role of the fatty acid oxygenase genes ppoA and ppoC in 1-octen-3-ol biosynthesis by A. luchuensis, we constructed ppoA and ppoC disruptants, ΔppoA and ΔppoC, respectively, via protoplast-PEG transformation. No clear differences in growth and conidiation were observed between the transformants and the parent strain. Volatile compounds in rice koji prepared using these gene disruptants were analyzed by SPME-GCMS. The amount of 1-octen-3-ol contained in koji produced by the ΔppoA strain was the same as that produced by the parental strain. In contrast, although the ΔppoC strain grew on the rice koji, 1-octen-3-ol was not detected. These results indicate that ppoC is involved in 1-octen-3-ol biosynthesis in A. luchuensis.

Generation of Trichoderma reesei Mutant with Enhanced Xylanase Activity by Using Disparity Mutagenesis.

In the current study, we attempted to enhance the xylanase activity of Trichoderma reesei ATCC66589 by using disparity mutagenesis, wherein a plasmid harboring proofreading-impaired DNA polymerase δ was inserted. Following selection on xylan-rich media and successive plasmid curing, a mutant showing conidiospores strikingly different from those of the parent strain, with many small humped-surface spheres, was generated. Xylanase and ß-xylosidase activities of the mutant XM1, cultivated in xylan medium, were 15.8- and 11.0-fold higher than those of the parent strain, respectively. Furthermore, xylanase activity was generated approximately 24 h in advance compared to that in the parent. In contrast, when cultivated in Avicel medium, its xylanase and ß-xylosidase activities were 0.14- and 0.33-fold, respectively, compared to those in the parent. Among the xylan component sugars and related polyols, D-xylose and xylobiose exerted a distinct inductive effect on the xylanase activity in Avicel media, while xylitol and L-arabinose did not. Mutagenesis involved in xylose catabolism is suggestive of changes at the gene transcription level. Although the induction mechanism remains unclear in details, disparity mutagenesis may be useful for obtaining T. reesei mutants with high xylanase activity.

Efficient Continuous Production of Lactulose Syrup by Alkaline Isomerization Using an Organogermanium Compound.

Lactulose, a keto-type disaccharide widely used in pharmaceuticals and functional foods, is produced by the isomerization of lactose. The organogermanium compound poly-trans-[(2-carboxyethyl) germasesquioxane] (Ge-132) is an effective reaction promoter for the conversion of lactose to lactulose because of its high affinity to ketoses. Herein, an effective method for the continuous production of lactulose syrup was developed using Ge-132 through the alkaline isomerization of lactose in a bench-scale plant. This plant carried out a continuous isomerization process using Ge-132, continuous two-step separation process for separating the sugar and Ge-132, a continuous purification and concentration processes for the lactulose syrup, and separation and purification processes for the recovery of Ge-132. In this bench-scale plant, lactulose-containing syrup (350 g/L lactulose, 92 g/L lactose, and 31 g/L galactose) was prepared. The syrup was produced at a rate of 37.7 mL/h, and the content of residual Ge-132 in the syrup was 2 mg/L. The separation process was a two-step separation system requiring an ordinary electrodialyzer and an electro deionizer, which allowed the separation of more than 99.6 % Ge-132 from the reaction mixture. Moreover, the majority of Ge-132 and sodium hydroxide were recovered through electrodialysis using a bipolar membrane. The proposed system is the first to represent the novel development of an effective continuous production system for lactulose-containing syrup on the basis of the use of organogermanium compounds and incorporation of the electrodialysis technology.

Production of rice straw hydrolysis enzymes by the fungi Trichoderma reesei and Humicola insolens using rice straw as a carbon source.

Rice straw was evaluated as a carbon source for the fungi, Trichoderma reesei and Humicola insolens, to produce enzymes for rice straw hydrolysis. The enzyme activity of T. reesei and H. insolens cultivated in medium containing non-treated rice straw were almost equivalent to the enzyme of T. reesei cultivated in Avicel medium, a form of refined cellulose. The enzyme activity of T. reesei cultivated in medium containing NH4OH-treated rice straw was 4-fold higher than enzyme from cultures grown in Avicel medium. In contrast, H. insolens enzyme from cultures grown in NH4OH-treated rice straw had significantly lower activity compared with non-treated rice straw or Avicel. The combined use of T. reesei and H. insolens enzymes resulted in a significant synergistic enhancement in enzymatic activity. Our data suggest that rice straw is a promising low-cost carbon source for fungal enzyme production for rice straw hydrolysis.

Complementary function of two transketolase isoforms from Moniliella megachiliensis in relation to stress response.

Two transketolase isogenes, MmTKL1 and MmTKL2, isolated from Moniliella megachiliensis were investigated for their roles in stress response and erythritol biosynthesis. The encoded proteins were highly homologous in amino acid sequence and domain structure. Two stress response elements (STREs) were found upstream of MmTKL1, while no STRE was found upstream of MmTKL2. In contrast, two Ap-1 elements were present upstream of MmTKL2, but none were detected upstream of MmTKL1. MmTKL2 partially complemented the aromatic amino acid auxotrophy of a Saccharomyces cerevisiae tkl1 deletion mutant, suggesting that at least one of the MmTKLs functioned as a transketolase in vivo. In response to short-term osmotic stress (20% glucose or 1.2 M NaCl) in Moniliella cells, MmTKL1 expression increased rapidly through the first 40 min before subsequently decreasing gradually, while MmTKL2 expression showed no significant change. In contrast, short-term oxidative stress (0.15 mM menadione) induced considerable increases in MmTKL2, while MmTKL1 expression remained low under the same conditions. Long-term osmotic stress (20% glucose) yielded increased expression of both genes starting at 12 h and continuing through 72 h. During either osmotic or oxidative stress, intracellular erythritol accumulation could clearly be correlated with the pattern of expression of either MmTKL1 or MmTKL2. These results strongly suggested that MmTKL1 is responsible primarily for the response to osmotic stress, while MmTKL2 is responsible primarily for the response to oxidative stress. Thus, we postulate that the two transketolase isoforms of M. megachiliensis play distinct and complementary roles in coordinating erythritol production in response to distinct environmental stresses.

Efficient Alkaline Isomerization of Lactose to Lactulose in the Presence of an Organogermanium Compound.

Lactulose, a disaccharide widely used in pharmaceuticals and functional foods, is produced by lactose isomerization. Lactose and lactulose have an aldose-ketose relationship. Less than 25 % conversion of lactose into lactulose is achieved using the Lobry de Bruyn-Alberda van Ekenstein transformation with heating, whereas the conversion is increased to 80 % by the addition of an approximately equimolar concentration of the organogermanium compound 3-(trihydroxygermyl)propanoic acid (THGP) to the reaction mixture. To further understand this phenomenon, in this study, we analyzed the affinity between THGP and sugar isomers using 1H nuclear magnetic resonance spectroscopy. For the dimethyl derivative of THGP with lactose and lactulose, the complex formation ratios at 0.1 M (1:1 mixing ratio) were 14 and 59 %, respectively, with complex formation constants of 1.8 and 43 M-1, respectively. The complex formation capacity was approximately 24-fold higher for lactulose than for lactose. Moreover, THGP is considered to protect lactulose from alkaline degradation, resulting in high production yield of lactulose. Therefore, we concluded that high affinity for the isomerization product may promote isomerization and that promotion of sugar isomerization using an organogermanium compound is an effective method for converting lactose to lactulose.

The relationship between the violet pigment PP-V production and intracellular ammonium level in Penicillium purpurogenum.

Penicillium purpurogenum is the fungus that produces an azaphilone pigment. However, details about the pigment biosynthesis pathway are unknown. The violet pigment PP-V is the one of the main pigments biosynthesized by this fungus. This pigment contains an amino group in a pyran ring as its core structure. We focused on this pigment and examined the relationship between intracellular ammonium concentration and pigment production using glutamine as a nitrogen source. The intracellular ammonium level decreased about 1.5-fold in conditions favoring PP-V production. Moreover, P. purpurogenum was transferred to medium in which it commonly produces the related pigment PP-O after cultivating it in the presence or absence of glutamine to investigate whether this fungus biosynthesizes PP-V using surplus ammonium in cells. Only mycelia cultured in medium containing 10 mM glutamine produced the violet pigment, and simultaneously intracellular ammonium levels decreased under this condition. From comparisons of the amount of PP-V that was secreted with quantity of surplus intracellular ammonium, it is suggested that P. purpurogenum maintains ammonium homeostasis by excreting waste ammonium as PP-V.

Two glycerol 3-phosphate dehydrogenase isogenes from Candida versatilis SN-18 play an important role in glycerol biosynthesis under osmotic stress.

Two isogenes of glycerol 3-phosphate dehydrogenase (GPD) from Candida versatilis SN-18 were cloned and sequenced. These intronless genes (Cagpd1 and Cagpd2) were both predicted to encode a 378 amino acid polypeptide, and the deduced amino acid sequences mutually showed 76% identity. Interestingly, Cagpd1 and Cagpd2 were located tandemly in a locus of genomic DNA within a 262 bp interval. To our knowledge, this represents a novel instance of isogenic genes relating to glucose metabolism. The stress response element (STRE) was found respectively at -93 to -89 bp upstream of the 5'end of Cagpd1 and -707 to -703 bp upstream of Cagpd2, indicating that these genes are involved in osmotic stress response. In heterologous expression using a gpd1Δgpd2Δ double deletion mutant of Saccharomyces cerevisiae, Cagpd1 and Cagpd2 transformants complemented the function of GPD, with Cagpd2 being much more effective than Cagpd1 in promoting growth and glycerol synthesis. Phylogenetic analysis of the amino acid sequences suggested that Cagpd1p and Cagpd2p are NADP(+)-dependent GPDs (EC 1.1.1.94). However, crude enzyme extract from Cagpd1 and Cagpd2 transformants showed GPD activity with only NAD(+) as cofactor. Hence, both Cagpd1p and Cagpd2p are likely NAD(+)-dependent GPDs (EC 1.1.1.8), similar to GPDs from S. cerevisiae and Candida magnoliae.

Efficient Conversion of D-Glucose to D-Fructose in the Presence of Organogermanium Compounds.

D-Glucose and D-fructose are isomers of commonly consumed monosaccharides. The ratio of conversion of D-glucose to D-fructose by glucose isomerase (xylose isomerase) is not more than 50 %. However, addition of an equimolar ratio of the organogermanium compound poly-trans-[(2-carboxyethyl)germasesquioxane] (Ge-132) or its derivative increases the conversion ratio to 80 %. In contrast, use of the Lobry de Bruyn-Alberda van Ekenstein transformation with heating results in a lower conversion ratio, less than 30 %, whereas addition of an equimolar concentration of Ge-132 or its derivative to this reaction mixture increases the ratio to 73 %. Therefore, in this study, we aimed to further analyze the affinity between organogermanium compounds (i.e., Ge-132 and its derivatives) and sugar using 1H-nuclear magnetic resonance (NMR) spectrometry. For the dimethyl derivative of Ge-132, the complex formation ratios at 0.25 M (mixing ratio 1:1) were 19 and 74 % for D-glucose and D-fructose, respectively. Additionally, the complex formation constants between monosaccharides and Ge-132 were 1.2 and 46 M-1 for D-glucose and D-fructose, respectively. The complex formation capacity was approximately 40-fold higher for D-fructose than for D-glucose. Therefore, we concluded that the high affinity for the product of isomerization may promote isomerization, and that promotion of sugar isomerization using organogermanium compounds is an effective method for conversion of D-glucose to D-fructose.

Generation of a glucose de-repressed mutant of Trichoderma reesei using disparity mutagenesis.

We obtained a novel glucose de-repressed mutant of Trichoderma reesei using disparity mutagenesis. A plasmid containing DNA polymerase δ lacking proofreading activity, and AMAI, an autonomously replicating sequence was introduced into T. reesei ATCC66589. The rate of mutation evaluated with 5-fluoroorotic acid resistance was approximately 30-fold higher than that obtained by UV irradiation. The transformants harboring incompetent DNA polymerase δ were then selected on 2-deoxyglucose agar plates with hygromycin B. The pNP-lactoside hydrolyzing activities of mutants were 2 to 5-fold higher than the parent in liquid medium containing glucose. Notably, the amino acid sequence of cre1, a key gene involved in glucose repression, was identical in the mutant and parent strains, and further, the cre1 expression levels was not abolished in the mutant. Taken together, these results demonstrate that the strains of T. reesei generated by disparity mutagenesis are glucose de-repressed variants that contain mutations in yet-unidentified factors other than cre1.

NP24 induces apoptosis dependent on caspase-like activity in Saccharomyces cerevisiae.

Tomato NP24 is a homolog of osmotin, a PR-5 protein from tobacco that can initiate apoptosis in yeast via PHO36 in the plasma membrane. We cloned and sequenced NP24 from tomato cv. Momotaro. Based on phylogenetic analysis, NP24 from Momotaro belonged to the Solanaceae clade. The amino acid sequence was identical to that of cv. Ailsa Craig including signal peptide, but the residues predicted to interact with the adiponectin receptor, ADIPOR, were slightly different from osmotin. Recombinant NP24 (rNP24) was expressed in a reductase-deficient mutant of Escherichia coli as host cell, and purified from cell extract by affinity chromatography. Purified rNP24 significantly inhibited growth of Saccharomyces cerevisiae wild-type spheroplasts. In contrast, growth of PHO36 deletion mutant (ΔIzh2) spheroplasts was not inhibited. Moreover, rNP24 induced significant activity of reactive oxygen species, caspase-like activity, and also nuclear fragmentation in wild-type spheroplast cells. These results demonstrated that rNP24 from Momotaro greatly influenced cell viability due to triggering apoptosis through PHO36. Notably, apoptosis induced by NP24 was caspase-like protease dependent.

PP-O and PP-V, Monascus pigment homologues, production, and phylogenetic analysis in Penicillium purpurogenum.

The production of pigments as secondary metabolites by microbes is known to vary by species and by physiological conditions within a single strain. The fungus strain Penicillium purpurogenum IAM15392 has been found to produce violet pigment (PP-V) and orange pigment (PP-O),Monascus azaphilone pigment homologues, when grown under specific culture conditions. In this study, we analysed PP-V and PP-O production capability in seven strains of P. purpurogenum in addition to strain IAM15392 under specific culture conditions. The pigment production pattern of five strains cultivated in PP-V production medium was similar to that of strain IAM15392, and all violet pigments produced by these five strains were confirmed to be PP-V. Strains that did not produce pigment were also identified. In addition, two strains cultivated in PP-O production medium produced a violet pigment identified as PP-V. The ribosomal DNA (rDNA) internal transcribed spacer (ITS) region sequences from the eight P. purpurogenum strains were sequenced and used to construct a neighbor-joining phylogenetic tree. PP-O and PP-V production of P. purpurogenum was shown to be related to phylogenetic placement based on rDNA ITS sequence. Based on these results, two hypotheses for the alteration of pigment production of P. purpurogenum in evolution were proposed.

Metabolic correlation between polyol and energy-storing carbohydrate under osmotic and oxidative stress condition in Moniliella megachiliensis.

Moniliella megachiliensis, the osmo-tolerant basidiomycetous yeast was found to accumulate intracellularly energy-storing carbohydrates (trehalose and glycogen) along with polyols (glycerol and erythritol) up to stationary growth phase. In trehalose-loaded cell, osmotic-stress resulted in the rapid generation of glycerol, and oxidative stress with menadione resulted in the rapid generation of erythritol. Under either of these conditions, the levels of the energy-storing carbohydrates were depleted, while little glucose uptake was observed. These results suggested that the intracellular pools of trehalose and glycogen were rapidly converted to glycerol in response to osmotic stress, and to erythritol in response to oxidative stress and altered redox balance. Expression of tps1 encoding trehalose synthetic enzymes paralleled trehalose accumulation in the cell during the culture in 2% glucose, in contrast, expression of tpp1 or tpp2 encoding trehalose-6-phosphate phosphatase was little increased under the same condition. Expression of tre (tre1/tre2) encoding trehalose hydrolase (trehalase) increased with time associated with depletion of trehalose during oxidative stress. From these results, we concluded that glycerol and erythritol, the compatible solutes in M. megachiliensis were metabolically interrelated to energy-storing carbohydrates such as trehalose or glycogen during conditions of osmotic or oxidative stress.

Construction of transformation system in Penicillium purpurogenum.

Penicillium purpurogenum attracts attention in the food industry and biomass degradation. We expressed green fluorescent protein (GFP) with pBPE, a novel vector, and constructed a transformation system for P. purpurogenum. The accumulation of GFP was confirmed by fluorescence microscopy. In future, this system may prove useful for the genetic modification of P. purpurogenum.

Two transaldolase isogenes from Moniliella megachiliensis behave in a different way depending on the stress class.

We cloned and sequenced two transaldolase genes from Moniliella megachiliensis, a microorganism known to produce a significant amount of erythritol under hyper-osmotic stress. The amino acid sequences encoded by these two genes (MmTAL1, MmTAL2) showed 72% homology to each other. An AP-1 (ap response element) associated with oxidative stress was found in the promoter region of MmTAL1, while four STREs (stress response element) associated with osmotic stress were found in the promoter region of MmTAL2. In early-stage cultivation (up to 2 h), MmTAL1 was specifically expressed in response to oxidative stress generated by the presence of 0.15 mM menadione; expression level 3-fold higher than before stress loading. MmTAL2 was expressed in response to osmotic stress caused by 1.2 M NaCl; expression level was 21-fold higher than stress-free control. Erythritol accumulated intracellularly under osmotic and oxidative stress, approximately 30-fold and 35-fold, respectively. We therefore concluded that M. megachiliensis selectively uses two isogenes and produces erythritol during early-stage response to stress, depending on the type of environmental stress.

Tomato FRUITFULL homologs regulate fruit ripening via ethylene biosynthesis.

Certain MADS-box transcription factors play central roles in regulating fruit ripening. RIPENING INHIBITOR (RIN), a tomato MADS-domain protein, acts as a global regulator of ripening, affecting the climacteric rise of ethylene, pigmentation changes, and fruit softening. Previously, we showed that two MADS-domain proteins, the FRUITFULL homologs FUL1 and FUL2, form complexes with RIN. Here, we characterized the FUL1/FUL2 loss-of-function phenotype in co-suppressed plants. The transgenic plants produced ripening-defective fruits accumulating little or no lycopene. Unlike a previous study on FUL1/FUL2 suppressed tomatoes, our transgenic fruits showed very low levels of ethylene production, and this was associated with suppression of the genes for 1-aminocyclopropane-1-carboxylic acid synthase, a rate-limiting enzyme in ethylene synthesis. FUL1/FUL2 suppression also caused the fruit to soften in a manner independent of ripening, possibly due to reduced cuticle thickness in the peel of the suppressed tomatoes.

Transcriptional regulation of fruit ripening by tomato FRUITFULL homologs and associated MADS box proteins.

The tomato (Solanum lycopersicum) MADS box FRUITFULL homologs FUL1 and FUL2 act as key ripening regulators and interact with the master regulator MADS box protein RIPENING INHIBITOR (RIN). Here, we report the large-scale identification of direct targets of FUL1 and FUL2 by transcriptome analysis of FUL1/FUL2 suppressed fruits and chromatin immunoprecipitation coupled with microarray analysis (ChIP-chip) targeting tomato gene promoters. The ChIP-chip and transcriptome analysis identified FUL1/FUL2 target genes that contain at least one genomic region bound by FUL1 or FUL2 (regions that occur mainly in their promoters) and exhibit FUL1/FUL2-dependent expression during ripening. These analyses identified 860 direct FUL1 targets and 878 direct FUL2 targets; this set of genes includes both direct targets of RIN and nontargets of RIN. Functional classification of the FUL1/FUL2 targets revealed that these FUL homologs function in many biological processes via the regulation of ripening-related gene expression, both in cooperation with and independent of RIN. Our in vitro assay showed that the FUL homologs, RIN, and tomato AGAMOUS-LIKE1 form DNA binding complexes, suggesting that tetramer complexes of these MADS box proteins are mainly responsible for the regulation of ripening.