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
1999-12-10
2001-07-24
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...
C435S254200, C435S483000
Reexamination Certificate
active
06265186
ABSTRACT:
TECHNICAL FIELD
The present invention is in the field of genetic modification of yeast using recombinant DNA techniques. The invention is more particularly concerned with the production of yeast cells incorporating into the genome multiple copies of a desired gene, as well as yeast cells so obtained. The invention is further concerned with the production of proteins, peptides and metabolites, by growing yeast cells so obtained under conditions conducive to the expression of the said desired gene, whereby the production of the protein, peptide or metabolite is achieved.
BACKGROUND ART
A known method of obtaining yeast strains harbouring multiple copies of a desired gene is in the use of multicopy plasmids. Plasmids, however have several disadvantages: (i) the expression level expressed in terms of the ratio of desired protein produced to copy number of the desired gene is generally lower for plasmids than for integrated copies of the desired gene; thus, plasmids impose a bigger metabolic burden to the cell than integrated copies, which may result in a penalty in terms of biomass accumulation; (ii) plasmids are prone to structural instability which may lead to loss of the desired gene from the plasmid or to a partial protein product; (iii) plasmids may give rise to segregational instability leading to plasmid loss. The latter especially occurs if high level expression is desired which imposes a strong metabolic burden to the cell.
To overcome mitotic instability associated with plasmids, integration of multiple copies of a desired gene has met with some success in yeast. Multiple copies of desired genes have been integrated at a single integration site in the genome, mostly in the ribosomal DNA of the yeast genome. Multicopy integration at a single locus is almost invariably achieved by the integration of multiple copies in tandem in the yeast genome; this easily leads to instability due to out-recombination of copies of the construct as a consequence of the presence of direct repeats flanking the construct containing the desired gene. The more copies that are found in tandem, the easier it leads to excision by out-recombination of single copies, thereby decreasing protein production levels to an unpredictable, but lower, level.
To overcome some of the instability, a system was devised to obtain integration of high copy numbers in the ribosomal DNA by using a deficient selection marker, for example wherein a weak promoter is cloned in front of the selection marker (EP 0 481 008). A first disadvantage is the need of selection markers in the production strain. Dominant antibiotic-resistance markers are less desirable from a regulatory standpoint whereas on the other hand auxotrophic markers are often not useful because they require mutant recipient host strains. Moreover, the presence of selection markers may also add to an unwanted complexity of the fermentation. Secondly, the ribosomal RNA encoding DNA locus is found in a nuclear organelle called the nucleolus, which is not an ideal location for obtaining optimal expression of a protein-encoding gene, as protein encoding genes are RNA-polymerase-II transcribed genes.
It is an object of the invention to provide for yeast cells having multiple copies of a desired gene integrated into the genome without some or all of the problems encountered with yeast cells known in the prior art.
SUMMARY OF THE INVENTION
The present invention provides for the first time, as isolated clone, a yeast cell comprising at least two copies of a desired gene integrated into its chromosomal genome, wherein said chromosomal genome comprises at least two DNA domains suitable for integration of one or more copies of said desired gene, which domains share substantial sequence homology and are non-ribosomal RNA encoding DNA domains, and wherein at least two of said substantially homologous non-ribosomal RNA encoding DNA domains incorporate at least one copy of the said desired gene.
Preferred according to the invention are yeast cells, wherein at least two of said substantially homologous non-ribosomal RNA encoding DNA domains suitable for integration of one or more copies of said desired gene incorporate two or more copies of the said desired gene, more preferably, wherein each substantially homologous non-ribosomal RNA encoding DNA domain suitable for integration of one or more copies of said desired gene incorporates an identical number of copies of the said desired gene. Said substantially homologous non-ribosomal RNA encoding DNA domains suitable for integration of one or more copies of said desired gene may be substantial duplicates of each other, preferably allelic forms of each other. A preferred yeast cell according to the invention is one which is a Kluyveromyces yeast cell, such as a
K. lactis
or
K. fragilis.
Preferred according to the invention are desired genes which are recombinant genes, meaning that at least some part of the desired gene has been manipulated using modern genetic engineering techniques. According to one embodiment the desired gene is a yeast gene. According to another embodiment the desired gene is a non-yeast gene. In any event, “gene” means a gene capable of being expressed when present in the yeast host of choice. The present invention is exemplified by a yeast gene coding for &bgr;-galactosidase (LAC4, from
K. lactis
) and a non-yeast gene coding for chymosin. The LAC4 gene is an intracellular gene, the chymosin gene actually codes for a preproprotein which is secreted as prochymosin, which can be autocatalytically activated to form the mature protein product chymosin. A gene coding for chymosin is herein understood to include all forms of DNA sequences which encode polypeptide that can be processed or matured into a protein with chymosin activity and thus specifically include cDNAs encoding prepro- and prochymosin. The desired gene may be a recombinant gene comprising a transcription promoter region, and optionally further a transcription termination region, functional in the said yeast cell, wherein at least one of said regions, or both are not naturally linked to the open reading frame coding for a protein or peptide. It will be clear that desired genes having regulatory regions that are by their nature already functional in the yeast cell of choice can be expressed in yeast cells in their native state.
According to one preferred embodiment the number of non-ribosomal RNA encoding DNA domains suitable for integration of one or more copies of said desired gene is two and the number of copies of said desired gene per domain is three, preferably at least three, more preferably at least four, more preferably at least five.
According to one embodiment the yeast cell according to the invention is a marker gene free yeast cell.
According to another embodiment a yeast cell is provided wherein said substantially homologous non-ribosomal RNA encoding DNA domains are LAC4 alleles.
The invention further provides for a method of producing a protein or peptide, comprising the step of growing a yeast cell according to any one of the preceding claims under conditions conducive to the production of said protein or peptide. Optionally, the said method further comprises the step of recovering the protein, peptide or metabolite from the cells and/or from the culture medium.
According to yet another embodiment a method is provided of obtaining a yeast cell capable of producing a desired protein or peptide comprising the steps of: (a) transforming a yeast cell having a genome which comprises at least two DNA domains suitable for integration of one or more copies of a desired gene, which domains share substantial sequence homology and are non-ribosomal RNA encoding DNA domains, with a DNA molecule comprising a desired gene which codes for the said protein or peptide, or a precursor thereof, and a region of homology with a said domain; (b) selecting or screening for cells having obtained at least one copy of the said desired gene integrated into at least one of said non-ribosomal RNA encoding DNA domains suitable for integration of one or
Noordermeer-Van Der Haak Adriana Cornelia Maria
Swinkels Bart Willem
Van Ooijen Albert Johannes Joseph
Davis Katharine F
DSM N.V.
McDonnell & Boehnen Hulbert & Berghoff
Schwartzman Robert A.
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