Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or...
Patent
1997-03-20
1998-10-13
Elliott, George C.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
4351721, 4352551, 4352552, 4352555, 4352556, C12Q 102, C12N 1500, C12N 116, C12N 118
Patent
active
058210385
DESCRIPTION:
BRIEF SUMMARY
This application is a 371 of PCT/FR95/01226 filed Sep. 22, 1995.
The present invention relates to modified yeasts having a cellular permeability not conforming to the original one, induced by genetic mutation.
Yeast is a model of eukaryotic cells and an excellent tool for studying the metabolic pathways, the cell cycle, their respective regulatory systems, the isolation of genes, the production of heterologous proteins and the like. Yeast may also be used for the biotransformation of molecules of interest or for the discovery of biologically active compounds by in vivo screening. The latter applications require the penetration of xenobiotics inside the yeast cells.
The penetration of these molecules, like their concentration, inside the yeast cells depends on different parameters regulating cell "permeability". These different parameters act mainly at three different levels in yeast, its cell wall, its plasma membrane and its detoxification mechanisms.
Thus, for a molecule to penetrate inside yeast cells and to undergo biotransformation therein or to exert its biological effect therein in the case of a screening, it has to cross the wall (essentially composed of glucans, mannoproteins and chitins) and the plasma membrane (composed of sterols, phospholipids and proteins), two biological barriers which may weakly or strongly block its penetration. To maintain a high concentration inside the cell, it should not undergo detoxification, for example by reexpulsion towards the external medium or by metabolism inside the cell.
The literature reports a whole series of genes involved in the biosynthesis, the constitution and/or the integrity of the wall, the membrane and at the level of the detoxification systems.
Various genes are involved in the synthesis of the wall, such as the series of genes comprising KRE (Hutchins, 1983; Boone, 1990; Meader, 1990; Hill, 1992; Roemer, 1991; Brown, 1993; Klis, 1994), KTR (Lussier, 1993; Klis, 1994) and MNN (Kanik, 1990; Klis, 1994; Ballou, 1982). It has been possible to isolate different wall mutants such as the mutants csd, chs, och, skn1, pmr1, pmt1, erd1, vgr1, vgr4, cal3, shc1, pkc1, bck1, mkk1, mkk2, mpk1, ppz1, ppz2, pmi1 (Klis, 1994). The genes DHS1 (Lee, 1994), SRB1 (Stateva, 1991), as well as the genes involved in the signalling pathway for protein kinase C which negatively controls the degradation of the wall by different glucanases such as BGL2 (Shimizu, 1994; Klis, 1994), are more particularly involved in the integrity of this wall.
The identity of a whole set of genes involved in the biosynthesis of ergosterol, which is a predominant form of fungal sterols, has also been identified. Sterols are membrane constituents. By way of representative of these genes, there may be mentioned more particularly the genes ERG10 (Dequin, 1988), ERG11 and ERG13 (Servouse, 1984), HMG1 and RMG2 (Basson, 1986), ERG12 (Oulmouden, 1988), ERG8 (Tsay, 1991), ERG19 (Mons, 1984), ID11 and ERG20 (Anderson, 1989), ERG9 (Fegueur, 1991), ERG1 (Hoegenauer, 1991), ERG18 (Karst, 1977), ERG7 and ERG17 (Karst, 1977), ERG16 (Kalb, 1987), ERG24 (Marcireau, 1992), ERG6 (Hardwick, 1994), ERG2 (Ashman, 1991), ERG3 (Arthington, 1991), ERG5 (Molzhan, 1972), ERG4 (Lai, 1994). The phospholipids are also membrane constituents. Various genes involved in the synthesis of phospholipids have been identified, such as CHO1 (Kiyono, 1987), CHO2 (Kodaki, 1991), PEM2 (Kodaki, 1987), CK11 (Hosaka, 1989), CCT1 (Tsukagoshi, 1987), EPT1 and CPT1 (Hjelmstad, 1991), INO1 (Dean-Johnson, 1989), PIS1 (Nikawa, 1987) and the like. Likewise, various genes involved in the synthesis of fatty acids have been identified. Acetyl-CoA carboxylase (ACC1 gene (Al-Fell, 1992)) makes it possible to produce malonyl CoA which will be used by fatty acid synthase (the genes FAS1 (Schwezer, 1986) and FAS2 (Mohamed, 1990)) to give fatty acids. The unsaturation(s) will be introduced by virtue of the action of the fatty acid desaturase encoded by the OLE1 gene (Stukey, 1990).
As regards more particularly the detoxification phenomenon, it has been
REFERENCES:
Lees et al. (1992) Genetics and molecular biology of the genes functioning late in the sterol biosynthetic pathway in Saccharomyces. ACS Symp. Ser. 497:246-259, 1992.
Roemer et al. (1933) SN1 and KRE6 define a pair of functional homologs encoding putative membrane proteins involved in beta-glucan synthesis. Mol. Cell. Biol. 13:4039-4048, Jul. 1993.
Lee et al. (1993) A pair of functionally redundant yeast genes (PPZ1 and PPZ2) encoding type 1-related protein phosphatases function within the PKC1-mediated pathway. Mol. Cell. Biol. 13:5843-5853, Sep. 1993.
Nakanishi-Shindo (1993) Structure of the N-linked oligosaccharides that show the complete loss of alpha-1,6-polymannose outer chain from och1, och1 mnn1, and och1 mnn1 alg3 mutanta of Saccharomyces cerevisiae. J. Biol. Chem. 268:26338-26345, Dec. 1993.
Fleer Reinhard
Marcireau Christophe
Scherman Daniel
Elliott George C.
Rhone-Poulenc Rorer S.A.
Schwartzman Robert
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