Keiji Kondo

Dietary isohumulones, the bitter components of beer, raise plasma HDL-cholesterol levels and reduce liver cholesterol and triacylglycerol contents similar to PPARα activations in C57BL/6 mice

The effects of dietary isohumulones, the main components accounting for the bitter taste of beer, on lipid metabolism were examined. Young female C57BL/6N mice were fed diets containing isomerized hop extract (IHE), which consists mainly of isohumulones. Administration of IHE with an atherogenic (high-fat and high-cholesterol) diet for 2 weeks resulted in a significant increase in plasma HDL-cholesterol (P<0.01), along with a concomitant reduction in the atherosclerosis index, an increase in liver weight and a decrease in body weight gain in a dose-dependent manner. When animals received IHE with either a cholesterol or a basal diet for 1 week, significant decreases in the liver content of cholesterol (P<0.01) and triacylglycerol (cholesterol diet, P<0.01) were observed. Quantitative analyses of hepatic mRNA levels revealed that IHE administration resulted in up-regulation of mRNA for acyl-CoA oxidase, acyl-CoA synthetase, hydroxymethylglutaryl-CoA synthetase, lipoprotein lipase and fatty acid transport protein, and down-regulation of mRNA for Apo CIII and Apo AI. Administration of purified isohumulones effectively resulted in the same changes as IHE. Administration of fenofibrate, an agonist for PPARalpha, with a cholesterol diet caused marked hepatomegaly, an increase in plasma HDL-cholesterol, a decrease in hepatic cholesterol content, and alterations in hepatic mRNA levels similar to those observed in mice given IHE. Taken together, these results suggest that the modulation of lipid metabolism observed in mice fed diets containing isohumulones is, at least in part, mediated by activation of PPARalpha.

Production of lycopene by the food yeast, Candida utilis that does not naturally synthesize carotenoid.

The Erwinia uredovora crtE, crtB, and crtI genes, which are responsible for the synthesis of carotenoid lycopene from farnesyl pyrophosphate, were expressed in Candida utilis under the control of the promoters and terminators derived from the C. utilis GAP, PGK, and PMA genes, respectively. The yeast transformant carrying the carotenoid biosynthesis genes produced 758 microg/g dry weight of lycopene along with 407 microg/g dry weight of phytoene in the stationary phase. It was observed in the C. utilis transformant that ergosterol content was decreased to 65% of that in the parent strain that accumulated 6.04 mg/g dry weight of ergosterol. It is therefore possible that the carbon flux for the ergosterol biosynthesis has been branched at farnesyl pyrophosphate to generate a new pathway for the lycopene production in this yeast transformant.

Regulation and evaluation of five methanol-inducible promoters in the methylotrophic yeast Candida boidinii.

We isolated the promoter regions of five methanol-inducible genes (P(AOD1), alcohol oxidase; P(DAS1), dihydroxyacetone synthase; P(FDH1), formate dehydrogenase; P(PMP20), Pmp20; and P(PMP47), Pmp47) from the Candida boidinii genome, and evaluated their strength and studied their regulation using the acid phosphatase gene of Saccharomyces cerevisiae (ScPHO5) as the reporter. Of the five promoters, P(DAS1) was the strongest methanol-inducible promoter whose strength was approximately 1.5 times higher than that of the commonly used P(AOD1) in methanol-induced cells. Although the expression of P(AOD1) and P(DAS1) was completely repressed by the presence of glucose, formate-induced expression of P(FDH1) was not repressed by glucose. Expression under P(PMP47), another methanol-inducible promoter, was highly induced by oleate. The induction kinetics of P(PMP47) and P(DAS1) revealed that methanol induces the expression of peroxisome membrane protein Pmp47, earlier than the expression of matrix enzyme dihydroxyacetone synthase (Das1p), and that this information is contained in the promoter region of the respective gene. This is the first report which evaluates several methanol-inducible promoters in parallel in the methylotrophic yeast.

Construction of Protease-deficient Candida boidinii Strains Useful for Recombinant Protein Production: Cloning and Disruption of Proteinase A Gene (PEP4) and Proteinase B Gene…

The yeast Candida boidinii PEP4 and PRB1 genes, encoding proteinase A (PrA) and proteinase B (PrB), respectively, have been cloned and their primary structures were analyzed. The open reading frames of the PEP4 gene (1263 bp encoding a protein of 420 amino acids) and the PRB1 gene (1683 bp encoding a protein of 560 amino acids) were found. The deduced amino acid sequences of PrA and PrB are very similar to Saccharomyces cerevisiae PrA and PrB (64% and 61% identities, respectively). Both PEP4 and PRB1 genes were disrupted in the C. boidinii genome by one-step gene disruption. The resultant pep4Δ and the pep4Δ prb1Δ strains lost protease activity when compared with the wild-type original strain. The constructed C. boidinii strains are expected to be useful hosts for heterologous protein production.

Coenzyme Q system in the classification of some ascosporogenous yeast genera in the families Saccharomycetaceae and Spermophthoraceae.

The Co-Q systems of 11 strains representing the generaSchwanniomyces, Lodderomyces, Lipomyces, Nematospora andMetschnikowia were determined. All the genera were characterized by the Q-9 system except for the genusNematospora with needle-shaped ascospores. The only species,Nem. coryli, was found to have the Q-6 system. These results are discussed from the taxonomic point of view.

Cis-acting elements sufficient for induction of FDH1 expression by formate in the methylotrophic yeast Candida boidinii

The FDH1 gene of Candida boidinii encodes an NAD+-dependent formate dehydrogenase, which catalyzes the last reaction in the methanol dissimilation pathway. FDH1 expression is strongly induced by methanol, as are the promoters of the genes AOD1 (alcohol oxidase) and DAS1 (dihydroxyacetone synthase). FDH1 expression can be induced by formate when cells are grown on a medium containing glucose as a carbon source, whereas expression of AOD1 and DAS1 is completely repressed in the presence of glucose. Using deletion analyses, we identified two cis-acting regulatory elements, termed UAS-FM and UAS-M, respectively, in the 5′ non-coding region of the FDH1 gene. Both elements were necessary for full induction of the FDH1 promoter by methanol, while only the UAS-FM element was required for full induction by formate. Irrespective of whether induction was achieved with methanol or formate, the UAS-FM element enhanced the level of induction of the FDH1 promoter in a manner dependent on the number of copies, but independent of their orientation, and also converted the ACT1 promoter from a constitutive into an inducible element. Our results not only provide a powerful promoter for heterologous gene expression, but also yield insights into the mechanism of regulation of FDH1 expression at the molecular level.

The structure of the menaquinones with a tetrahydrogenated isoprenoid side-chain.

Menaquinones with a tetrahydrogenated isoprenoid side-chain of Oerskovia turbata and Brevibacterium lipolyticum were cyclized to the chromenyl acetate derivatives, which were then submitted to ozonolysis, followed by reduction with dimethylsulfide. The mass-spectrometric analyses of the ozonolysis products revealed the ion peaks at m/e 464 (M+), 449, 422, 407, 267 and 225. These results suggest that the two saturated double bonds are located continuously in the second and third units of the chain starting from the quinone ring, and the menaquinones are designated as 2-methyl-3-II,III-tetrahydromultiprenyl-1,4-naphthoquinone.

Carbohydrate Metabolism by the Acetic Acid Bacteria Part V:On the Vitamin Requirements for the Growth

Vitamin requirements for the growth of the acetic acid bacteria were investigated extensively on a. taxonomical viewpoint and the following new findings were pointed out. Neither Acetobacter nor Intermediate strain required vitamin for the growth.Gluconobacter required generally pantothenic acid. And some strains belonging to it did moreover somewhat of thiamine, nicotinic acid and p-aminobenzoic acid, although there was a difference of requirements between strains even in the same species. Riboflavin, pyridoxine, vitamin B12, folic acid, biotin and inositol were unnecessary for the growth of the acetic acid bacteria. A taxonomical division of the acetic acid bacteria based on the vitamin requirements agreed well with that on basis of the oxidative activities for carbohydrates.

Identification and application of novel autonomously replicating sequences (ARSs) for promoter-cloning and co-transformation in Candida utilis.

In order to develop practical recombinant DNA techniques in the industrially important yeast Candida utilis, at least six plasmids harboring autonomously replicating sequences (ARSs) were isolated from a C. utilis genomic library. Two ARSs were subjected to detailed analysis. Sequences of 1.9 and 1.8 kb were found to be necessary to exert ARS activity in a plasmid as assessed by transformation efficiency and mitotic stability. Both fragments were found to be rich in AT content (69.5% and 70.8% respectively), and to contain an 11-bp ARS consensus sequences (10 and 13 motifs with one base difference respectively). Using the ARS-containing plasmid as a promoter-cloning vector, several DNA fragments having promoter activities were cloned and characterized. Co-transformation of C. utilis with an integrating DNA fragment and a replicating plasmid yielded plasmid-free transformants harboring the fragment integrated into the C. utilis genome.

Substrate recognition mechanism of a glycosyltrehalose trehalohydrolase from Sulfolobus solfataricus KM1.

Glycosyltrehalose trehalohydrolase (GTHase) is an α‐amylase that cleaves the α‐1,4 bond adjacent to the α‐1,1 bond of maltooligosyltrehalose to release trehalose. To investigate the catalytic and substrate recognition mechanisms of GTHase, two residues, Asp252 (nucleophile) and Glu283 (general acid/base), located at the catalytic site of GTHase were mutated (Asp252→Ser (D252S), Glu (D252E) and Glu283→Gln (E283Q)), and the activity and structure of the enzyme were investigated. The E283Q, D252E, and D252S mutants showed only 0.04, 0.03, and 0.6% of enzymatic activity against the wild‐type, respectively. The crystal structure of the E283Q mutant GTHase in complex with the substrate, maltotriosyltrehalose (G3‐Tre), was determined to 2.6‐Å resolution. The structure with G3‐Tre indicated that GTHase has at least five substrate binding subsites and that Glu283 is the catalytic acid, and Asp252 is the nucleophile that attacks the C1 carbon in the glycosidic linkage of G3‐Tre. The complex structure also revealed a scheme for substrate recognition by GTHase. Substrate recognition involves two unique interactions: stacking of Tyr325 with the terminal glucose ring of the trehalose moiety and perpendicularly placement of Trp215 to the pyranose rings at the subsites −1 and +1 glucose.