Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Patent
1998-01-06
2000-07-04
Horlick, Kenneth R.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
435 71, 435 699, 4352542, C12P 2106, C12P 2104, G01N 3353, C12N 116
Patent
active
060837179
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to a system allowing for improved protein expression in yeasts, more particularly in Yarrowia lipolytica. It relates to recombinant upstream activator sequences, promoter sequences and more specifically hybrid promoter sequences, functional in Yarrowia, and vectors containing them. Another object for the invention is a recombinant yeast transformed with such vectors, and a process for producing a protein in Yarrowia lipolytica.
Yarrowia lipolytica is a dimorphic yeast which can grow on a limited number of carbon sources, including glucose and glycerol, paraffines such as n-alkanes and alkenes (Klug and Markovetz, 1967 and 1969; Bassel and Mortimer, 1973), lipids and proteins (Ogrydziak et al., 1977) but not sucrose. To degrade proteins, depending on growth conditions, Yarrowia lipolytica secretes proteases (Ahearn et al., 1968, Kamada et al., 1972). For example, in alkaline or neutral medium, it secretes an Alkaline Extracellular Protease (AEP) (Tobe et al., 1976; Ogrydziak et al., 1977; Ogrydziak and Scharf, 1982). The corresponding gene, XPR2, has been cloned independently by Davidow et al. (1987), Matoba et al. (1988) and Nicaud et al. (1989).
Many eukaryotic promoters, especially yeast promoters, have now been extensively studied. It has been demonstrated that regulation of gene expression involves multiple interactions between transcription factors bound within a promoter. Multiple sites may be required for, and multiple proteins may be associated with, the functioning of even the smallest cis-acting elements. In yeast cells, upstream activation sequences (UAS.sub.S) are necessary for transcription. They function in either orientation and at a variable distance with respect to the TATA box and transcription start site, but in contrast to enhancers in higher eukaryotes, they must be upstream from these basal elements. UAS.sub.S are targets for two types of trancriptional activators. The first class of activators, which includes HAP1 and ACE1, bind UAS.sub.S only under conditions of active transcription of the downstream gene. The second class is represented by ADR1, HSTF, PUT3, and GAL4; these proteins are permanently bound to UAS.sub.S, but their activity is regulated.
Although most repression phenomena in yeast cells seem to result from inactivation or absence of transcription factors, some negative regulatory sites, or upstream repression sequences (URS), have also been identified.
The XPR2 gene from Yarrowia lipolytica encodes an inducible alkaline extracellular protease (AEP) which is the major protein secreted by this yeast (Davidow et al., 1987). The regulation of this gene is complex. Derepression of AEP occurs at pH above 6.0 on media lacking preferred carbon and nitrogen sources; full induction of the XPR2 promoter requires high levels of peptones in the culture medium, although the exact nature of the inducer is unknown. The functional XPR2 promoter encompasses roughly 900 bp.
EP 138 508 discloses a process for transformation of Yarrowia lipolytica by integration of DNA having a region homologous with chromosomal DNA and a genetic marker.
EP 220 864 discloses a vector for expression and secretion of heterologous proteins in Yarrowia lipolytica. The vector must comprise a promoter sequence of a Yarrowia lipolytica gene and the signal sequence of the XPR2 gene of Yarrowia lipolytica operably linked to a gene for a heterologous protein. The promoter sequence can be the XPR2 promoter sequence, ie the upstream untranscribed region in front of the signal sequence.
Surprisingly, the inventors have shown that hybrid promoters containing only parts of the XPR2 promoter sequence can be used to obtain expression of a protein in Yarrowia lipolytica.
Furthermore, they have shown that hybrid promoters composed of tandem repeats of only a part of the XPR2 promoter sequence will allow the strong quasi-constitutive expression of a protein under its control, irrespective of the presence of peptones in the medium. These hybrid promoters are no longer repressed by the preferred car
REFERENCES:
S. Blanchin-Roland et al., "Two upstream activation sequences control the expression . . . Yarrowia lipolytica", Mol. Cell. Bio. 14(1), 1994, 327-338.
C. Gaillardin et al., "Genetic engeineering in Yarrowia lipolytica", J. Bas. Micro., vol. 28, No. 3 pp. 161-174, 1988.
Davidow et al. J. of Bact. vol. 169, No. 1 pp. 4621-4629, 1987.
Heslot et al. Micro Appl. Food Biotech abstract 1 sheet, 1990.
Blanchin-Roland Sylvie
Gaillardin Claude
Madzak Catherine
Horlick Kenneth R.
Institut National Agronomique Paris- Grignon
Institut National de la Recherche Agronomique
Siew Jeffrey
LandOfFree
Upstream activator sequences and recombinant promoter sequences does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Upstream activator sequences and recombinant promoter sequences , we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Upstream activator sequences and recombinant promoter sequences will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-1485091