Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Enzymatic production of a protein or polypeptide
Patent
1984-01-18
1987-04-14
Wiseman, Thomas G.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Enzymatic production of a protein or polypeptide
4351723, 435317, 435255, 935 28, 935 56, 935 69, 935 11, C12P 2100, C12N 1500, C12N 100
Patent
active
046578579
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to the microbiological preparation of preprothaumatin and related compounds, which preparation has been made possible by recombinant DNA techniques. In European patent applications (A2) 0 054 330 and 0 054 331, both published on June 23, 1982, DNA sequences encoding various allelic forms of preprothaumatin, their maturation forms such as prethaumatin, prothaumatin and thaumatin, and modified forms thereof, as well as cloning vehicles comprising said DNA sequences, their use in transforming micro-organisms, in particular E. coli, and the preparation of thaumatin like proteins are described.
For the production to be commercially attractive, the yields obtained with E. coli are not high enough, so that a need exists for microbiological systems which are capable of producing the thaumatin-like proteins in far higher amounts. By thaumatin-like proteins are meant preprothaumatin, its various allelic or modified forms, and their maturation forms such as prethaumatin, prothaumatin and thaumatin.
It has now been found that the structural genes described in the above-mentioned European patent applications can be combined with autonomously replicating sequences originating from Kluyveromyces lactis (so-called KARS), with a selection marker and with a yeast regulon into a plasmid, which plasmid can be brought into yeast cells of the Kluyveromyces genus, which then become capable of producing the thaumatin-like proteins. In this way expression of the thaumatin-like proteins in yeast cells succeeded.
Therefore, the present invention provides a yeast of the genus Kluyveromyces containing a recombinant DNA plasmid comprising:
(i) a structural gene encoding for preprothaumatin or its various allelic or modified forms or their maturation forms,
(ii) an autonomously replicating sequence originating from Kluyveromyces lactis (so-called KARS),
(iii) a yeast regulon derived from the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes of Saccharomyces cerevisiae, and
(iv) a selection marker, such as the trp-1 or the lac-4 gene.
In this way a much improved expression of the desired proteins could be obtained, so that commercial application comes more within reach.
Preferably the plasmid also contains a transcription terminator derived from a yeast, and/or a centromer and/or a replicating region derived from a yeast.
Good results have been obtained with a Kluyveromyces lactis yeast, but it is very probable that other Kluyveromyces species will be effective as well. The structural genes are preferably selected from the group consisting of the structural genes disclosed in the above-mentioned European patent applications.
Successful KARS-sequences were isolated from the wild strain K. lactis CBS 2360, as will be described below.
For transformation purposes in K. lactis it is desirable to use selectable markers on the vectors, for example the tryptophan gene (trp-1) and the lactase gene (lac-4) which originate from S. cerevisiae and K. lactis, respectively. These DNA-sequences are effective not only as selective markers, but also in enabling a selective pressure to be exerted on the system during further propagation.
Preferably the transcription terminator, if used, is selected from the group consisting of a terminator sequence originating from the Hind III-Eco RI fragment of the 2 micron DNA vector of S. cerevisiae and a terminator sequence derived from the GAPDH genes of S. cerevisiae.
The presence of a centromer or replicating region derived from a yeast chromosome, e.g. from S. cerevisiae or from K. lactis improves the stability of the plasmids according to the invention.
The invention further provides a process for preparing a yeast of the genus Kluyveromyces as described above, which process comprises the preparation of a recombinant DNA plasmid by combining a structural gene as indicated above with a KARS-sequence and upstream of the structural gene with a yeast GAPDH regulon and with a selection marker, and optionally with a centromer or replicating region derived from a yeast chromosome and o
REFERENCES:
Edens et al, 1982, "Cloning of CDNA Encoding the Sweet-Tasting Plant Thaumatin and its Expression in E. coli", Gene, v 18, pp. 1-12.
Struhl et al, 1979, "High-Frequency Transformation of Yeast: Autonomous Replication of Hybrid DNA Molecules", Proc Natl Acad Sci, v 76, 1035-1039.
Edens et al, 1984, "Synthesis and Processing of the Plant Protein Thaumatin in Yeast", Cell, v 37, pp. 629-633.
Holland et al, 1980, "Structural Comparison of Two Nontandemly Repeated Yeast Glyceraldehyde-3-Phosphate Dehydrogenase Genes", J. Biol. Chem, v 255, 2596-2605.
Cover of Gene, v 18 No. 1, Apr. 1982.
Edens Luppo
Ledeboer Adrianus
van den Berg Johannes A.
Verrips Cornelis
Giesser Joanne M.
Internationale Octrooi Maatschappij "Octropa" B.V.
Wiseman Thomas G.
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