Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Patent
1994-01-13
1997-06-03
Ketter, James S.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
4351723, 435183, 435189, 4352542, 43525421, C12P 2102, C12N 115
Patent
active
056353693
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
The present invention relates to a yeast strain in the chromosomes of which several heterologous genes are stably integrated. The present invention relates, in addition, to a method of expression in yeast of a complex function linked to the activity of several heterologous factors, and especially to a method of expression in yeast of the monooxygenase activity of heterologous cytochrome(s) P450.
BACKGROUND OF THE INVENTION
The cytochromes P450 (hereinafter abbreviated to P450) constitute a superfamily of membrane enzymes having very varied monooxygenase type activities. Their activities are capable of being used in a wide range of fields of application. There may be mentioned, by way of non-limiting examples: by means of reactions of insertion of an oxygen atom, followed or otherwise by rearrangements in carbon--carbon or carbon-hydrogen bonds, and by means of the addition of oxygen to a variety of hetero atoms (sulfur, nitrogen, phosphorus). P450 utilizes aerial oxygen as oxidizing agent. human hepatic metabolism of natural or artificial xenobiotic molecules (pollutants, medicinal products, additives). Such prediction is of primary importance, especially in the case of the development of new molecules of pharmaceutical importance. the environment.
In view of this broad field of application, the expression of heterologous forms of P450 and of their activities in microorganisms which lack them clearly merits attention. Two problems then became apparent: first, the membrane nature of the P450 of eukaryotes makes it desirable to use a host microorganism of the eukaryotic type, leading to the choice of yeasts; second, these enzymes are functional only in the presence of "associated enzymes" which play the part of specific electron transporters. Whereas the diversity of cytochromes P450 is extreme, the "associated REDOX enzymes" are few in number and comprise cytochrome b5, NADH-cytochrome b5 reductase and, most especially, NADPH-cytochrome P450 reductase. Moreover, certain enzymes, termed phase II, such as, for example, microsomal epoxide hydrolase, may be needed for the metabolic coupling between different P450's, or for the destruction of highly reactive chemical intermediates formed during certain reactions, such as, for example, the metabolism of polycyclic hydrocarbons.
The high level in vivo expression of heterologous cytochromes P450 in yeast leads to high levels of activity only if these "associated redox enzymes", which are essential to the functioning of P450, can be coexpressed in the yeast, in suitable stoichiometries relative to one another and to the P450. Recent data show that, activity as a result of the lack of reductase. of a considerable fraction of the P450 as a result of the large increase in the number of abortive catalytic cycles and of the production by the excess of reductase of oxygen-containing radicals which are dangerous to the cell. The outcome is a loss of viability of the cells, or even a drop in activity due to the destruction of cytochromes P450. class B P450. Moreover, the presence of a high level of cytochrome b5 appears to exert a protective effect against the toxic effects linked to excessive reductase levels; P450 activity, and can potentially reduce the amount of unbound intracellular heme and thus be toxic to the cell.
In order to solve these problems, there was constructed according to the invention, by genomic integration of synthetic genes, a set of yeast strains expressing, stably and in a manner which can be modulated (by the composition of the culture medium), an enzymatic environment which can be optimized for expression of the activity of any heterologous P450.
Many publications have described the expression of heterologous cytochromes P450 in yeast. Nevertheless, the problem of optimization of the cellular environment to permit high activity in vivo has seldom been tackled, the proteins produced often being used for analytical purposes or purposes of in vitro research.
The problem of optimization of the activities has been tackled hit
REFERENCES:
patent: 5312735 (1994-05-01), Fink et al.
Urban et al., Biochimie, vol. 72, 1990, pp. 463-472.
Cullin, C. and Pompon, D. "Synthesis of functional mouse cytochromes P-450 P1and chimeric P450 P3-1 in the yeast Saccharomyces cerevisiae," Gene, vol. 65, pp. 203-217 (1988).
Cullin Christophe
Pompon Denis
Slonimski Piotr
Truan Gilles
Urban Philippe
Centre National de la Recherche Scientifique
Ketter James S.
LandOfFree
Yeast strains with stable integration of heterologous genes does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Yeast strains with stable integration of heterologous genes, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Yeast strains with stable integration of heterologous genes will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-390664