DNA coding for extracellular glucoamylase genes STAI and STA3 was isolated from DNA libraries of two Saccharomyces diastaticus strains, each carrying STAI or STA3. Cells transformed with a plasmid carrying either the STAI or STA3 gene secreted glucoamylases having the same enzymatic and immunological properties and the same electrophoretic mobilities in acrylamide gel electrophoresis as those of authentic glucoamylases. Southern blot analysis of genomic DNA from S. diastaticus and a glucoamylase-non-secreting yeast, Saccharomyces cerevisiae, revealed that the STAI and STA3 loci of S. diastaticus showed a high degree of homology, and that both yeast species (S. diastaticus and S. cerevisiae) contained DNA segments highly homologous to those of the extracellular glucoamylase genes. Restriction maps of the homologous DNA segments suggested that the extracellular glucoamylase genes of S. diastaticus may have arisen from recombination among the resident DNA segments in S. cerevisiae.
Extracellular glucoamylase produced by a starch-fermenting yeast, Saccharomyces diastaticus 5106-9A, was purified. The enzymewas found to be heterogeneous in molecular weight, ranging from approximately 80K to 66K as estimated by gel filtration, and consisted of two subunits, H and Y. The molecular weight of subunit H was heterogeneous and was determined to be approximately 68K, 59K, and 53K by acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The molecular weight of subunit Y was 14K, estimated by the same gel. the molecular weight of the deglycosylated form of subunit H was 4IK, suggesting that the heterogeneity of the enzyme was due to glycosyl moieties of subunit H. Subunits H and Y were separated by gel filtration in the presence of sodium dodecyl sulfate. Subunit Y seemed to be hydrophobic, since it was insoluble in an aqueous buffer without detergent. Saccharomyces diftstaticus carrying any one of the unlinked STA genes (STA1, STA2 and STA3) produces extracellular glycoamylases This investigation was supported in part by a Grant-in-Aid for Scientific Research (Grant No. 57211018) from the Ministry of Education, Science and Culture of Japan. Added in proof. Molecular weight of deglycosylated subunit Y was determined to be 3.4K in SDS-urea gel electrophoresis according to the method of Swankand Mankres.
The carbazole-3,4-quinone alkaloids, carquinostatin A (1) 1) and lavanduquinocin (2) 2) were isolated from Streptomyces exfoliates 2419-SVT2 and Streptomyces viridochromogenes by Seto and co-workers in 1993 and 1995, respectively. The structures of the two alkaloids were elucidated to be the same carbazole-3,4-quinone moiety by NMR spectral analyses and other spectroscopic experiments. The absolute stereochemistry of the C-11 position of the two alkaloids was the same R-configuration. Carquinostatin A (1) and lavanduquinocin (2) were also shown to be a potent neuronal cell protecting substance which exhibits a free radical scavenging activity. Total syntheses of these alkaloids have recently been developed by the Kn枚lker group. [3][4][5][6][7][8] The transition metalmediated and -catalyzed methodologies for the construction of the carbazole framework have been efficiently employed.Throughout the course of this study, we have been interested in the synthetic development of biologically active condensed-heteroaromatic compounds, including natural products, by the thermal electrocyclic reactions of either conjugated hexatriene [9][10][11][12][13][14][15][16][17] or monoazahexatriene 18) systems incorporating one double bond from an aromatic or heteroaromatic portion. We recently reported the synthesis of the highly substituted carbazole alkaloids, carazostatin 9) and carbazoquinocins, 9,10) by the construction of the appropriate carbazole framework based on the allene-mediated electrocyclic reaction of the 6p-electron system involving the indole 2,3-bond. In the present paper, we describe the asymmetric synthesis of 6-desprenyl-carquinostatin A (6-descycloavandulyllavanduquinocin) 3, which is a common carbazole framework of both alkaloids, based on a lipase-catalyzed esterification using a racemic alcohol 6 for the determination of the absolute stereochemistry of 3. We chose the 3-ethoxy-2-methyl-1-(trifluoromethylsulfonyloxy)carbazole (4), 9,10) as a starting material, which was prepared in a six-step sequence from 3-iodoindole-2-carbaldehyde by the application of our methodology, as shown in the retrosynthetic Chart 1.The required 1-allylcarbazole 5 was prepared from the triflate 4 and allyboronic acid pinacol ester in the presence of PdCl 2 (dppf) in dimethylformamide (DMF) by the SuzukiMiyaura reaction. 19,20) An asymmetric synthesis of the core carbazole structure, 6-desprenyl-carquinostatin 3 and 6-descycloavandulyl-lavanduquinocin 3, toward a total synthesis of carquinostatin A (1) and lavanduquinocin (2)
In recent times, demands for bio-oils, such as biolipids and biofuels, have progressed, since they are very significant biomolecules for human health and for the ecological preservation of the earth's environment. Nevertheless, the supplements of bio-oils were not always stable because the harvests of oleaginous plants, animals, and fishes were influenced by climates and marine conditions. Various research and developments were carried out on microbial production of valuable oils, such as polyunsaturated fatty acids, from the viewpoint of factory-controllable fermentative formation of specified products. So far, it has been reported on the production of eicosapentaenoic acid (Shimizu et al., 1988), docosahexaenoic acid (Li and Ward, 1994), and g-linolenic acid (Hansson and Dostalek, 1988) using various oleaginous fungi. These products, however, were entirely localized at the inner space of cell. Therefore processes for cell disruption and oil extraction were essential to recover such products as other oleaginous plants or animals. The development of extracellular accumulation of fats (triacylglycerols, TGs) must be promising for an improvement in the cost performance of microbial lipid fermentation.Previously we reported the breeding of mutant strains of yeast Candida lipolytica that secrete palmitic acid into glucose medium (Miyakawa et al., 1984), and of the yeast Trichosporon sp., which accumulates TGs in the ethylpalmitate (Yagi et al., 1994) or glucose (Nojima et al., 1995) medium. As far as we know, no other reports exist concerning the extracellular production of useful lipids by microorganisms. We could exhibit the construction of lipid-secretable microorganisms, though their productivities were economically insufficient. For further improvement of the capacity to secrete lipid, it would be necessary to have not only a screening of a superior producer, but also an analysis of the molecular mechanism for the outward flow of lipids. We would advocate that the extracellular accumulation of hydrophobic biomaterials by the fermentation process has a higher probability to be realized if the equilibrium of membrane transport for hydrophobic compounds could be made up to incline toward pumping out. It is widely known that the yeast Saccharomyces cerevisiae is one of the most widely studied microorganisms in molecular genetics. Studies on lipid-excreting mechanisms using a mutant strain of S. cerevisiae should have advantages to breed useful microorganisms for lipid-accumulating fermentation. In this paper we describe isolation and characterization of the S. cerevisiae mutant strain that secretes TG into glucose medium. Moreover, we represent compleIsolation and characterization of triacylglycerol-secreting mutant strain from yeast, Saccharomyces cerevisiae Department of Bioscience and Biotechnology, Faculty of Engineering, Fukuyama University, Fukuyama 729-0292, Japan (Received September 11, 1998; Accepted January 7, 1999) To establish the molecular bases for development of a microbiological system approach...
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