A novel DNA microarray method to detect one line of genetically modified (GM) soybean and five lines of GM maize was developed using multiplex PCR coupled with primer extension on a plastic plate. Multiplex PCR products were applied on an extension primer-immobilized plate and the spots corresponding to the DNA sequences were visualized. This method is a rapid and simple way to detect GM soybean and GM maize optically.

View full abstract

The inhibitory effect of (−)-, (+)-matairesinol and (−)-, (+)-secoisolariciresinol on the discoloration of dark muscle (chiai in Japanese) of two-year-old yellowtail (hamachi in Japanese) was evaluated by measuring the X and a^* values. (−)-Matairesinol was most effective for retaining the red color of dark muscle in this experiment.

View full abstract

The core structure of the telomerase inhibitor, dictyodendrin B, was synthesized by using the palladium-catalyzed cross-coupling reaction of 3-aryl-1-(2-arylethyl)-4-hydroxy-2,5-bismethoxycarbonylpyrrole triflate with 7-alkoxyindole-3-boronate as the key step.

View full abstract

The relationship between the stereochemistry and antimicrobial activity of butane-type lignans was clarified. All stereoisomers of dihydroguaiaretic acid (DGA) showed both antibacterial and antifungal activity. The (+)- and (−)-7,7′-dioxodihydroguaiaretic acid (ODGA) also showed both antibacterial and antifungal activity, while meso-ODGA did not show antibacterial activity, but showed antifungal activity. No activity of any stereoisomer of secoisolariciresinol (SECO) was apparent.

View full abstract

Stereochemically restricted analogues of C-7 substituted 7-epi-jasmonate, together with 12-hydroxy jasmonic acid, 12-hydroxy jasmonic acid glucoside, and jasmonic acid conjugated with L-isoleucine (JA-Ile), were synthesized and then tested for potato tuber cell expansion-inducing activity. JA-Ile showed almost the same activity as JA, while the C-7 substituted 7-epi-jasmonates exhibited weaker activity than JA and showed an antagonist effect against JA.

View full abstract

Volicitin [N-(17-hydroxylinolenoyl)-L-glutamine] has previously been identified from the lepidopteran larval regurgitant as an elicitor of plant volatile emission. The efficient incorporation of free oxygen into volicitin by Spodoptera litura larvae is demonstrated here by rearing them under ¹⁸O₂ for three days. ¹⁸O-labeling of the hydroxyl group of volicitin was confirmed by liquid chromatography/mass spectrometry-ion trap-time-of-flight (LC/MS-IT-TOF) and suggests the activity of a monooxygenase in volicitin biosynthesis.

View full abstract

A new cyclohexenone compound was isolated as a mixture of enantiomers from a culture filtrate of Lasiodiplodia theobromae. The relative structure was determined to be 4,5-dihydroxy-3-methyl-cyclohex-2-enone on the basis of MS, ¹H-NMR, and ¹³C-NMR spectroscopic analyses, including 2D-NMR experiments. Resolution of the enantiomers was conducted by a coupling reaction with (S)-MTPA-Cl followed by HPLC separation.

View full abstract

The enantioselective synthesis of aspergillide B, a 14-membered macrocyclic cytotoxin, was achieved in a 49% yield via 7 steps from a synthetic intermediate of aspergillide C. The spectroscopic data and specific rotation value for the synthetic material matched those of natural aspergillide B.

View full abstract

An enzyme that catalyzes the stoichiometric conversion of 6-tuliposide into tulipalin was purified and characterized from bulbs of Tulipa gesneriana. The enzyme appeared to be a dimer, the relative molecular mass (Mr) of each subunit being 34,900; it had maximum activity and stability at neutral pH and moderate temperature. The enzyme preferentially acted on such glucose esters as 6-tuliposides, and to a lesser extent on p-nitrophenylacetate.

View full abstract

In the course of our chemical screening program for novel metabolites by LC-MS monitoring, we isolated a new aspochracin derivative, JBIR-15 (1), together with aspochracin, from the culture broth of a sponge-derived fungus, Aspergillus sclerotiorum Huber Sp080903f04. The structure of 1 was determined to be N-demethyl aspochracin at the alanyl residue on the basis of extensive NMR and MS analyses.

View full abstract

Four compounds were isolated from scrap cultivation beds of the mushroom, Hericium erinaceum. Compounds 1–4 were identified as methyl 4-hydroxy-3-(3-methylbutanoyl) benzoate, 2-chloro-1,3-dimethoxy-5-methylbenzene, methyl 4-chloro-3,5-dimethoxybenzoate, and 4-chloro-3,5-dimethoxybenzaldehyde by an interpretation of the NMR and MS data, respectively. This is the first reported isolation of 1 from a natural source. All the compounds showed protective activity against endoplasmic reticulum stress-dependent cell death.

View full abstract

Denervation is known to induce skeletal muscle atrophy and fiber-type transitions, the molecular mechanisms of which are poorly understood. To investigate the effect of denervation on skeletal muscle, proteomic analysis was performed to compare denervated soleus muscle with normal soleus muscle. The muscles were fractionated to myofibrillar and sarcoplasmic fractions, which were analysed using two-dimensional gel electrophoresis (2-DE), followed by MALDI-TOF-MS. At least 30 differentially regulated proteins were identified in the sarcoplasmic fractions of normal and denervated soleus muscles. This group included metabolic enzymes, signaling molecules, chaperones, and contractile proteins. We also found two proteins, APOBEC-2 (RNA-editing enzyme) and Gamma-synuclein (breast cancer related protein), which have not been recognized as denervation-induced proteins to date. Our results might prove to be beneficial in elucidating the molecular mechanisms of denervation-induced muscle atrophy.

View full abstract

Diabetes frequently develops in Huntington’s disease patients. Here, we found that mutant huntingtin forms aggregates in the cytoplasm and reduces insulin secretion from huntingtin transfected pancreatic β cell lines, NIT-1 cells. Activity of the pro-survival factor, Akt, is enhanced in these cells, which might improve the maintenance of insulin content. Overexpression of heat shock protein 40 (HSP40) inhibits aggregation, reverses impaired insulin release, and blocks the enhancement of Akt activity. These results suggest that impairment of β cells is mostly linked with the aggregate formation of mutant huntingtin, and that HSP40 ameliorates the malfunction of pancreatic β cells by inhibiting aggregation.

View full abstract

Two genes, encoding YK-LiP1 and YK-LiP2, were cloned from the white-rot fungus Phanerochaete sordida YK-624, and a homologous expression system for the gene was constructed. Two full-length cDNAs (ylpA and ylpB) were isolated by degenerate RT-PCR and RACE-PCR. The results of N-terminal amino acid sequence analysis of native YK-LiP1 and YK-LiP2 showed that ylpA and ylpB coded for YK-LiP2 and YK-LiP1 respectively. The promoter of glyceraldehyde-3-phosphate dehydrogenase cloned from P. sordida YK-624 (PsGPD) was used to drive the expression of ylpA. Expression vector pGPD-g-ylpA was transformed into a P. sordida YK-624 uracil auxotrophic mutant, UV-64. The YlpA protein was secreted in active form by the transformants after 4 d of growth in a medium containing an excessive nitrogen source, whereas endogenous YK-LiP1 and YK-LiP2 were not produced. The physical and catalytic properties of the purified YlpA protein were very similar to those of YK-LiP2. These results suggest that homologous expression of recombinant YK-LiP2 was successful.

View full abstract

8S-Lipoxygenase (8S-LOX) is known as a mouse homolog of human 15S-LOX-2. 15S-LOX-2 was down-regulated in malignant transformation of prostate epithelial cells, and its overexpression caused cell cycle arrest. To determine whether 8S-LOX would have a growth inhibitory effect on prostate carcinoma, we obtained human prostate carcinoma PC-3 cells expressing 8S-LOX or 15S-LOX-2. The growth rate of cells measured by colorimetric assay was reduced by expression of 8S-LOX and 15S-LOX-2. The addition to enzyme-expressing cells of arachidonic acid enhanced the growth suppressive effect, whereas the expression of catalytically inactive mutants did not affect cell growth, suggesting that the effect was product-dependent. DNA microarray and quantitative reverse transcription-PCR analyses revealed that the c-myc mRNA coding region determinant-binding protein/insulin-like growth factor II mRNA-binding protein 1 (CRD-BP/IMP-1), known as an oncofetal protein, was down-regulated in 8S-LOX- and 15S-LOX-2-expressing PC-3 cells. Targeted knockdown of CRD-BP/IMP-1 resulted in inhibition of the DNA synthesis rate of PC-3 cells as measured by bromodeoxyuridine incorporation. We propose that expression of 8S-LOX and 15S-LOX-2 suppresses CRD-BP/IMP-1 expression, resulting in inhibition of human prostate carcinoma PC-3 cell proliferation.

View full abstract

We isolated a homolog of cathepsin D from the cDNA library of the olive flounder kidney. The olive flounder cathepsin D transcript consisted of 1,733 bp that encoded a polypeptide of 396 amino acids. The overall similarity between olive flounder cathepsin D and other cathepsin Ds was very high, with the highest amino acid sequence identity to barramundi perch (89%). RT-PCR revealed that cathepsin D was expressed in almost all tissues, with high expression in the liver, intestine, kidney, skin, and spleen. The accumulation of cathepsin D mRNA after bacterial infection, as determined by RT-PCR, was constitutive and increased greatly after bacterial infection.

View full abstract

Psilocybe argentipes is a hallucinogenic mushroom. The present study examined the effects of P. argentipes on marble-burying behavior, which is considered an animal model of obsessive-compulsive disorder. P. argentipes significantly inhibited marble-burying behavior without affecting locomotor activity as compared with the same dose of authentic psilocybin. These findings suggest that P. argentipes would be efficient in clinical obsessive-compulsive disorder therapy.

View full abstract

Previous studies have shown that the muscle protein degradation rates of broiler are lower than those of layer chickens. In this study, we assessed proteolytic-related gene expression in the pectoralis muscle of layer and broiler chickens. The mRNA levels of atrogin-1/MAFbx and proteasome C2 subunit, but not those of ubiquitin, m-calpain large subunit, cathepsin B, or caspase-3, were lower in the skeletal muscle of the broilers than in the layers at 7 and 14 d of age. We infer that the lower muscle protein degradation of broilers than of layers at least partly relates to lower mRNA expression of atrogin-1/MAFbx and proteasome C2 subunit in the skeletal muscle of broilers.

View full abstract

OsWRKY53, a chitin oligosaccharide elicitor-responsive rice WRKY gene, has been found to be involved in defense responses in rice. We identified three tandem W-box elements, putative recognition sites for WRKY transcription factors, as cis elements that are essential to the elicitor-responsiveness of OsWRKY53 by deletion and mutation analysis of the promoter by dual luciferase assay.

View full abstract

The relative drying rate of samples (RDR), which is the ratio of the drying rate of pretreated samples to that of untreated ones, might be used as a tool to investigate the damage to cells of agroproducts induced by high-pressure treatment. Damage to cells induced by high pressure was estimated by comparing the RDR after high-pressure pretreatment with the RDR after chloroform-vapor, heat, and freeze-thaw pretreatments of Japanese radish samples. The RDR after high-pressure pretreatment was similar to the RDR after chloroform-vapor pretreatment, and was lower than for heat, and for freeze-thaw pretreatment. For agroproducts, high-pressure treatment is thus comparatively moderate.

View full abstract

Rice bran contains various polyphenolic compounds with anti-oxidative activities, and it has long been known to inhibit melanogenesis, but the inhibition mechanism has not been fully elucidated. Cofermentation of rice bran with Lactobacillus rhamnosus and Saccharomyces cerevisiae significantly reduced the cytotoxicity of the resulting extract to B16F1 melanoma cells. Marked reduction of α-melanocyte stimulating hormone (MSH) induced melanin synthesis was also observed upon treatment with fermented rice bran extract but it had no direct inhibitory effect on tyrosinase activity, while the intracellular tyrosinase activity was reduced by the extract. This result was further confirmed by an immunoblot assay measuring the level of tyrosinase protein. In addition, the expression of microphthalmia-associated transcription factor (MITF), a key regulator of melanogenesis, was significantly decreased by the extract. All together, the fermented rice bran extracts showed an inhibitory effect on melanogenesis through downregulation of MITF, along with reduced cytotoxicity.

View full abstract

Two groups of Chinese hypertriacylglycerolemic subjects were recruited and randomized to medium- and long-chain triacylglycerols (MLCT) oil or long-chain triacylglycerols (LCT) oil. Two subgroups were divided by age at less or more 60 years in both groups. Both oils were consumed at 25–30 g daily for 8 weeks. Anthropometry, blood biochemicals, and computed tomography (CT) scanning were done at the initial and final times. In subjects of age less than 60 years on MLCT, the body weight, body mass index (BMI), waist circumference (WC), hip circumference (HC), waist-hip ratio (WHR), body fat, total fat area, and subcutaneous fat area were significantly lower than those of the initial values, and the change values in these indicators and visceral fat area lowered significantly as compared with those on LCT. The levels of apoB, apoA2, apoC2, and apoC3 decreased significantly, and the change in values in the levels of triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), apoA1, apoB, apoA2, apoC2, apoC3 were significantly lower on MLCT of age under 60 years as compared with those on LCT.

View full abstract

We optimized the isolation protocol for intraepithelial lymphocytes (IELs) and lamina propria lymphocytes (LPLs) from the rat small intestine, and LPLs from even the rat large intestine. The major population of IELs in the small intestine was considered to be from the villus epithelia. The cytotoxicity of mucosal leukocytes was comparable among isolated fractions from both the small and large intestines, regardless of the population differences. Further analyses of the cells collected from other lymphoid tissues demonstrated that CD161⁺ cells selectively accumulated in the intestinal lamina propria and did not recirculate through the lymph ducts. Our modified isolation protocol enables the collection of mucosal immune cells from the rat intestines without any deterioration of cell function and could contribute to a better understanding of dietary influences on the mucosal immune system.

View full abstract

To assess the digestion and assimilation of gelatin and gelatin hydrolysates, the in situ absorption of typical hydroxyproline-containing dipeptides, Pro-Hyp, Hyp-Gly, Ser-Hyp Ala-Hyp, and pentadecapeptide, (Pro-Hyp-Gly)₅, was investigated in the rat small intestine. During vascular perfusion after the injection of Hyp-Gly, Pro-Hyp and (Pro-Hyp-Gly)₅ into the jejunum, peptide-form Hyp but not free-Hyp gradually increased in the perfusate. In contrast, in the case of Ser-Hyp and Ala-Hyp, both free- and peptide-form Hyp rapidly increased. The presence of these dipeptides and the pentadecapeptide in the perfusates was confirmed by liquid chromatography-tandem mass spectrometry (LC-MS/MS), using multiple reaction monitoring (MRM). Some digestive and absorbed forms from (Pro-Hyp-Gly)₅ were identified as Gly-(Pro-Hyp-Gly)₄, (Pro-Hyp-Gly)₄, Gly-(Pro-Hyp-Gly)₃, (Pro-Hyp-Gly)₃, Gly-(Pro-Hyp-Gly)₂, and (Pro-Hyp-Gly)₂ by MALDI-TOF/MS. The dipeptide hydrolase activity in intestinal mucosa toward Pro-Hyp and Hyp-Gly was extremely low, while Ser-Hyp and Ala-Hyp were substantially hydrolyzed in the cytosol. These results suggest that Hyp-peptides were resistant to intracellular hydrolysis and that a significant amount of these peptides was transported across the intestinal wall and may enter the portal circulation in an intact form.

View full abstract

Ganoderma tsugae is a medicinal fungus with several biological activities. It has long been used as a folk remedy for the promotion of health and longevity in China and other oriental countries. Here, a bioactive fraction of G. tsugae was progressively purified to be enriched in the activity of cytoprotective enzymes. The highest bioactivity was detected in the 20% EtOH-precipitated fraction, which was prepared from submerged fermentation filtrate of G. tsugae. Following further purification by gel filtration chromatography and acetone extraction, the most bioactive fraction, F5-2, was identified as a peptidoglycan-like compound. Extracts of G. tsugae (F5-2) induced heme oxygenase-1 (HO-1) and thioredoxin reductase-1 (TrxR1) expression in endothelial cells by increasing NF-E2-related factor-2 (Nrf2) nuclear translocation. Pretreatment with F5-2 increased intracellular glutathione (GSH) and protected against H₂O₂, suggesting that induction of these antioxidant enzymes is important in protection against oxidative stress. Hence the bioactive peptidoglycan-like compound from G. tsugae might protect endothelial cells.

View full abstract

We examined the effect of a crude hot-water extract (HW) of quince (Cydonia oblonga Miller) fruit on type I allergy in vivo and in vitro. The oral administration of the quince HW-added diet to NC/Nga mice for 63 d showed a significant decrease in the development of atopic dermatitis-like skin lesions under conventional conditions. The concentration of IgE in the serum collected from mice fed with quince HW was also lowered in a dose-dependent manner. Moreover, we found that quince HW inhibited the release of β-hexosaminidase from rat basophilic leukemia cell line RBL-2H3 after a 24-h treatment. The quince HW fraction of less than 3 kDa reduced the mRNA expression of the high-affinity IgE receptor (FcεRI) γ subunit. These results suggest that quince HW had an inhibitory effect on type I allergy by suppressing IgE production and IgE-mediated degranulation.

View full abstract

The health promoting effects of a grapevine-shoot extract named Vineatrol^®30, which contains resveratrol (Resv) as well as considerable amounts of Resv oligomers, have recently been investigated. In the present study, we analyzed the free radical scavenging capacity, the ability to inhibit lipid peroxidation, and the capacity to enhance the human glutathione peroxidase 1 (GPx) and the human superoxide dismutase 1 (SOD) gene promoter activities of Vineatrol^®30. Vineatrol^®30 was able to scavenge the 2,2′-azinobis-3-ethylbenzothiazoline-6-sulfonic acid radical cation and led to concentration-dependent inhibition of lipid peroxidation, Vineatrol^®30 not being superior to Resv alone in both cases. Vineatrol^®30 also enhanced the gene promoter activities of human GPx and SOD expressed in V79 cells, whereas this effect could not be demonstrated for Resv. In summary, the results presented in this study show that the Vineatrol^®30 grapevine-shoot extract is a free radical scavenger and potent antioxidant at non-cytotoxic concentrations.

View full abstract

Acetic acid (AcOH), a main component of vinegar, recently was found to suppress body fat accumulation in animal studies. Hence we investigated the effects of vinegar intake on the reduction of body fat mass in obese Japanese in a double-blind trial. The subjects were randomly assigned to three groups of similar body weight, body mass index (BMI), and waist circumference. During the 12-week treatment period, the subjects in each group ingested 500 ml daily of a beverage containing either 15 ml of vinegar (750 mg AcOH), 30 ml of vinegar (1,500 mg AcOH), or 0 ml of vinegar (0 mg AcOH, placebo). Body weight, BMI, visceral fat area, waist circumference, and serum triglyceride levels were significantly lower in both vinegar intake groups than in the placebo group. In conclusion, daily intake of vinegar might be useful in the prevention of metabolic syndrome by reducing obesity.

View full abstract

Flavor properties are important factors of soybean seeds in their utilization as food materials. In order to isolate novel varieties and mutants of soybean having preferable flavor properties, a simple and efficient screening system was established using an automated headspace sampler coupled to gas chromatography. With this system, five major volatile compounds were analyzed within 12 min. By applying this screening system to 626 soybean varieties collected worldwide, we isolated four soybean varieties that showed unique compositions of volatile compounds. Through biochemical analysis, it was found that the uniqueness of three of them was possibly independent of lipoxygenase enzyme, and thus perhaps this screening system can expand the subject of flavor properties beyond lipoxygenase and thus be useful in discovering new types of soybeans.

View full abstract

Studies of the physiological functions of intestinal epithelial cells (IECs) have been limited by the difficulty of primary culture of IEC. We established a method for primary culture of mouse IEC by culturing fragments of fetal small intestines pretreated with EDTA. This method reproducibly resulted in the expansion of cytokeratin-positive epithelial cells, and vigorous expansion of the epithelial cells was observed only from intestinal fragments of embryonic days 15-16. These cells expressed alkaline phosphatase activity and major histocompatibility complex (MHC) class II molecules, indicating the mature phenotype of IEC in a small intestine. The cells also presented antigens to CD4⁺ T cells. Furthermore, the cells expressed various cytokines and chemokines, and the expression was enhanced by bacterial stimulation. These results indicate that the primary-cultured mouse IEC prepared by the method established here can be a beneficial tool in study of the functions of IECs, especially in mucosal immunity.

View full abstract

Biliary excretion of quercetin metabolites in conscious rats was biphasic, a high peak within 3 h and a lower peak for next 6 h, after duodenal administration of soluble quercetin glycosides. We also found much higher biliary excretion of quercetin metabolites than urinary excretion on feeding a quercetin glycoside diet. These results suggest high levels of enterohepatic circulation of quercetin metabolites.

View full abstract

Two compounds were isolated from biotransformation of daidzein, a soybean isoflavone, by Aspergillus oryzae. One was 6,7,8,4′-tetrahydroxyisoflavone, an A-ring dihydroxylated daidzein, and the other was a novel compound which had the cleaved B-ring of daidzein. The former was perhaps derived from 8-hydroxydaidzein and/or 6-hydroxydaidzein, and had high DPPH radical-scavenging activity.

View full abstract

Zerumbone (ZER), a sesquiterpene in Zingiber zerumbet Smith, has been implicated as a promising chemopreventive agent. Here we found that ZER suppressed expression of pro-inflammatory genes (COX-2 and iNOS) and induced detoxification genes (GSTP1 and NQO1) in RAW264.7 macrophages. Using the ZER-bound Sepharose gel, it appeared ZER was covalently bound to proteins, Keap1 and HuR, that play key roles in these molecular mechanisms.

View full abstract

We constructed a LongSAGE (Long Serial Analysis of Gene Expression) library from a 3-d culture of Phanerochaete chrysosporium supplemented with atropine, which inhibits the production of lignin-degrading enzymes. The library (the atropine library) contains 13,108 LongSAGE tags and 6,783 unique tags. The gene expression profile represented by the tags was compared with those of two previously constructed libraries, one of which was constructed using 2-d cultures in which the fungus had not yet produced ligninolytic enzymes (the 2-d library) and the other was constructed using 3-d cultures in which the fungus had just started to produce the enzymes (the 3-d library). We found a total of 595 genes that were at least twice more highly or at least twice less highly expressed in the 3-d library than in the 2-d library or the atropine library, and the fluctuations were statistically significant. The relationships among these 595 genes were considered using cluster analysis. Of the 595 genes, 164 showed expression patterns similar to those of four ligninolytic enzyme genes, which were more expressed on day 3 than under any other conditions. Many of these 164 genes comprised genes possibly involved in lignin degradation, lipid metabolism, xenobiotic degradation, stress response, or signal transduction pathways.

View full abstract

The APSES protein family includes important transcriptional regulators of morphological processes in ascomycetes. We identified a deletion mutant of the APSES protein Mstu1 in Magnaporthe grisea that showed reduced conidiation and mycelial growth. Mstu1 formed a number of appressoria comparable to the wild type, although appressorium formation was delayed. In M. grisea, rapid transfer of conidial glycogen and lipid droplets to incipient appressoria is required for appressorial turgor generation, which the fungus uses to penetrate plant cuticles. Appressorial turgor was low in mstu1 and the mutant was deficient in appressorium-mediated invasion of rice leaves. The transfer of conidial glycogen and lipid droplets was remarkably delayed in mstu1, and a consequent delay in degradation of these conidial reserves was observed. Our results indicate that Mstu1 is required for appressorium-mediated infection due to its involvement in the mobilization of lipids and glycogen.

View full abstract

Gluconobacter sp. NBRC3259 converted glycerol to glyceric acid (GA). The enantiomeric composition of the GA produced was a mixture of DL-forms with a 77% enantiomeric excess of D-GA. After culture conditions, such as initial glycerol concentration, types and amounts of nitrogen sources, and initial pH, were optimized, Gluconobacter sp. NBRC3259 produced 54.7 g/l of GA as well as 33.7 g/l of dihydroxyacetone (DHA) from 167 g/l of glycerol during 4 d of incubation in a jar fermentor with pH control. GA production from raw glycerol samples, the main by-product of the transesterification process in the biodiesel production and oleochemical industries, was also evaluated after proper pretreatment of the samples. Using a raw glycerol sample with activated charcoal pretreatment, 45.9 g/l of GA and 28.2 g/l of DHA were produced from 174 g/l of glycerol.

View full abstract

Polylactic acid is receiving increasing attention as a renewable alternative for conventional petroleum-based plastics. In the present study, we constructed a metabolically-engineered Candida utilis strain that produces L-lactic acid with the highest efficiency yet reported in yeasts. Initially, the gene encoding pyruvate decarboxylase (CuPDC1) was identified, followed by four CuPDC1 disruption events in order to obtain a null mutant that produced little ethanol (a by-product of L-lactic acid). Two copies of the L-lactate dehydrogenase (L-LDH) gene derived from Bos taurus under the control of the CuPDC1 promoter were then integrated into the genome of the CuPdc1-null deletant. The resulting strain produced 103.3 g/l of L-lactic acid from 108.7 g/l of glucose in 33 h, representing a 95.1% conversion. The maximum production rate of L-lactic acid was 4.9 g/l/h. The optical purity of the L-lactic acid was found to be more than 99.9% e.e.

View full abstract

Two vectors, pT7NScamAB and pRED, have been used for the functional expression of bacterial class I cytochrome P450 (P450) genes in Escherichia coli, which utilize putidaredoxin reductase (CamA) and putidaredoxin (CamB), and the reductase domain of a self-sufficient P450RhF respectively, for electron transfer from NAD(P)H to a P450 protein. We here compared the efficiency of bioconversion with the two vectors towards n-octane, cyclohexane, n-butylbenzene, and 2-n-butylbenzofuran using two well-characterized CYP153A genes, aciA and CYP153A13a (P450balk). As for n-octane bioconversion, aciA and pT7camAB was the best combination for the production of 1-octanol and 1,8-octanediol. As for the bioconversion of cyclohexane, n-butylbenzene and 2-n-butylbenzofuran, CYP153A13a with pRED achieved the most efficient bioconversion, as compared by conversion ratio per active CYP153A protein content. It was also found that 2-n-butylbenzofuran is biotransformed into 4-benzofuran-2-yl-butyric acid via 4-benzofuran-2-yl-butan-1-ol with E. coli cells expressing CYP153A.

View full abstract

A novel retrotransposon Rhizot was identified in Rhizopus oryzae and R. delemar. Rhizot has a unique structure that consists of a pol ORF similar to non-LTR (long terminal repeat) retorotransposons between two LTRs. Rhizot was distributed in all Rhizopus species tested. The Rhizot pol gene was transcribed in the liquid culture, and was induced by UV and oxidative stress.

View full abstract

To determine whether two HorC molecules coordinately form a single unit, the functional properties of covalently linked dimers of HorC encoded by tandemly fused horC genes were studied. Lactobacillus brevis introduced with the fused horC genes and a single horC gene exhibited same degree of resistance to hop compounds and cetyltrimethylammonium bromide. This suggests that HorC functions as a homodimer.

View full abstract