Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1998-09-23
2001-01-30
Schwartzman, Robert A. (Department: 1636)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S091100
Reexamination Certificate
active
06180363
ABSTRACT:
The present invention relates to the recoding of DNA sequences which encode proteins which contain regions having a high content of codons which are poorly translated by yeasts, in particular which encode proteins of plant origin, such as the P450 cytochromes of plant origin, and to their expression in yeasts.
It is known that certain sequences encoding proteins of interest, in particular proteins of plant origin, are not readily translated in yeasts. This applies, in particular, to proteins which possess regions having a high content of codons which are poorly suited to yeasts, in particular leucine codons, such as some P450 cytochromes of plant origin. Some systems which have been developed for improving the expression of P450 cytochromes of animal or plant origin in yeasts, such as those described by Pompon et al. (
Methods Enzymol
., 272, 1996, 51-64; WO 97/10344), have turned out to be unsuitable for large numbers of P450 cytochromes which encompass regions having a high content of codons which are poorly suited to yeasts.
The P450 cytochromes constitute a superfamily of membrane enzymes of the monooxygenase type which are able to oxidize a large family of generally hydrophobic substrates. The reactions are most frequently characterized by the oxidation of C—H or C═C bonds, and of heteroatoms, and, more rarely, by the reduction of nitro groups or by dehalogenation. More specifically, these enzymes are involved in the metabolism of xenobiotic substances and drugs and in the biosynthesis of secondary metabolites in plants, some of which have organoleptic or pharmacodynamic properties.
As a consequence, the P450 cytochromes are used, in particular, in:
the in vitro diagnosis of the formation of toxic or mutagenic metabolites (molecules of natural origin, pollutants, drugs, pesticides, etc.), making it possible, in particular, to develop novel active molecules (pharmaceutical, agrochemistry),
the identification and destruction of molecules which are toxic for, or pollute, the environment,
the enzymic synthesis of novel molecules.
The search for heterologous expression of P450 cytochromes by host cells, more specifically yeasts, is therefore important for obtaining controlled production of this enzyme in large quantity, either for isolating it and using it in the above-listed processes, or for using the transformed cells directly for the said processes without previously isolating the enzyme.
The present invention provides a solution to the abovementioned problem, enabling proteins which contain regions having a high content of codons which are poorly suited to yeasts, in particular P450 cytochromes of plant origin, to be expressed in yeasts.
The present invention therefore relates to a DNA sequence, in particular a cDNA sequence, which encodes a protein of interest which contains regions having a high content of codons which are poorly suited to yeasts, characterized in that a sufficient number of codons which are poorly suited to yeasts is replaced with corresponding codons which are well-suited to yeasts in the said regions having a high content of codons which are poorly suited to yeasts.
Within the meaning of the present invention, “codons which are poorly suited to yeasts” are understood as being codons whose frequency of use by yeasts is less than or equal to approximately 13 per 1000, preferably less than or equal to approximately 12 per 1000, more preferably less than or equal to approximately 10 per 1000. The frequency at which codons are used by yeasts, more specifically by
S. cerevisiae
, is described, in particular, in “Codon usage data base from Yasukazu Nakamura” (http://www.dna.affrc.go.jp/~nakamura/codon.html). This applies, in particular, to codons CTC, CTG and CTT, which encode leucine, to codons CGG, CGC, CGA, CGT and AGG, which encode arginine, to codons GCG and GCC, which encode alanine, to codons GGG, GGC and GGA, which encode glycine, and to codons CCG and CCC, which encode proline. The codons which are poorly suited to yeasts in accordance with the invention are, more specifically, codons CTC and CTG, which encode leucine, CGG, CGC, CGA, CGT and AGG, which encode arginine, codons GCG and GCC, which encode alanine, GGG and GGC, which encode glycine, and codons CCG and CCC, which encode proline.
Within the meaning of the present invention, “corresponding codons which are well-suited to yeasts” are understood as being the codons which correspond to the codons which are poorly suited to yeasts and which encode the same amino acids, and whose frequency of use by yeasts is greater than 15 per 1000, preferably greater than or equal to 18 per 1000, more preferably greater than or equal to 20 per 1000. This applies, in particular, to codons TTG and TTA, preferably TTG, which encode leucine, to codon AGA, which encodes arginine, to codons GCT and GCA, preferably GCT, which encode alanine, to codon GGT, which encodes glycine, and to codon CCA, which encodes proline.
Within the meaning of the present invention, “region having a high content of codons which are poorly suited to yeasts” is understood as being any region of the DNA sequence which contains at least 2 poorly suited codons among 10 consecutive codons, with it being possible for the two codons to be adjacent or separated by up to 8 other codons. According to one preferred embodiment of the invention, the regions having a high content of poorly suited codons contain 2, 3, 4, 5 or 6 poorly suited codons per 10 consecutive codons, or contain at least 2 or 3 adjacent poorly suited codons.
Within the meaning of the present invention, “sufficient number of codons” is understood as being the number of codons which it is necessary and sufficient to replace in order to observe a substantial improvement in their expression in yeasts. Advantageously, at least 50% of the codons which are poorly suited to yeasts in the high-content region under consideration are replaced with well-suited codons. Preferably, at least 75% of the poorly suited codons of the said region are replaced, with 100% of the poorly suited codons more preferably being replaced.
Within the meaning of the present invention, “substantial improvement” is understood as being either a detectable expression when no expression of the reference sequence is observed, or an increase in expression as compared with the level at which the reference sequence is expressed.
Within the meaning of the present invention, “reference sequence” designates any sequence which encodes a protein of interest and which is modified in accordance with the invention in order to promote its expression in yeasts.
The present invention is particularly well suited to DNA sequences, in particular cDNA sequences, which encode proteins of interest which contain regions having a high content of leucine and in which a sufficient number of CTC codons encoding leucine in the said region having a high content of leucine is replaced with TTG and/or TTA codons, or in which a sufficient number of CTC and CTG codons encoding leucine in the said region having a high content of leucine is replaced with TTG and/or TTA codons, preferably with a TTG codon.
Within the meaning of the present invention, “region having a high content of leucine” is understood as being a region which contains at least 2 leucines among 10 consecutive amino acids in the protein of interest, with it being possible for the two leucines to be adjacent or separated by up to 8 other amino acids. According to one preferred embodiment of the invention, the regions having a high content of leucine contain 2, 3, 4, 5 or 6 leucines per 10 consecutive amino acids, or contain at least 2 or 3 adjacent leucines.
According to a preferred embodiment of the invention, at least 50% of the CTC or CTC and CTG codons of the region having a high content of leucine are replaced with TTG or TTA codons, with at least 75% of the CTC or CTC and CTG codons of the said region preferably being replaced, and 100% of the CTC or CTC and CTG codons more preferably being replaced.
Advantageously, the present invention is particularly suitable for DN
Batard Yannick
Durst Francis
Schalk Michel
Werck-Reichhart Daniele
Botts Baker
Davis Katharine F
Rhone-Poulenc Agro
Schwartzman Robert A.
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