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
2001-07-17
2003-10-28
Ketter, James (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...
C435S320100, C435S461000, C435S254230, C435S255500, C435S255600, C536S023100, C536S023200
Reexamination Certificate
active
06638735
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to expression cassettes for transforming
Pichia ciferrii
. More particularly, it relates to expression cassettes containing
Pichia ciferrii
ribosomal DNA fragment, CYH
r
gene resistant to cycloheximide, and a desired gene, and to uses thereof.
2. Description of the Prior Art
Pichia ciferrii
has been used to biologically desulfurize coals (Stevens et al., U.S. Pat. No. 4,851,350), to produce D-alpha-amino acids (Takelchi et al., U.S. Pat. No. 5,068,187), to produce (S)-1-phenyl-1,3-propandiol (Ajinomoto, JP 6-90789A) or to produce secondary alcohols by stereospecific ketone reduction (Merck, EP-300287). Further, it produces and secretes tetraacetyl phytosphingosine (TAPS) which is a precursor of ceramides (Barenholz et al., Biochem. Biophys. Acta, 248, 458, 1971; ibid, 306, 341, 1973).
Phytosphingosines including TAPS, like ceramides, show an activity of surface skin-protection and of preventing excessive water-loss and dry out of the skin, facilitating their uses in cosmetics. They can be obtained from various microorganisms and easily converted to ceramides by N-acylation.
TAPS productions by wild type
Pichia ciferrii
ATCC 14091 and F-60-10 (NRRL 1301) are not satisfactory for commercial uses. To improve the production of TAPS in the strains of
Pichia ciferrii
, attempts to provide mutants which are capable of producing a higher level of TAPS have been made (Wickerham & Burton, J. Bacteriol., 80, 484, 1960; U.S. Pat. No. 5,618,706). The present inventors also developed novel useful mutant (KFCC-10937) which allows a larger amount of TAPS production in a shorter time (KR 98-49305A).
Pichia ciferrii
had been classified into genus Hansenula and is recently reclassified into genus Pichia by 5S-RNA analysis (Yamada et al.,
Biosci, Biotechnol. Biochem
., 58, 1245, 1994). By this reason, the genetic study of the Pichia yeasts is not sufficient and transformation method of
Pichia ciferrii
has not been established.
The present invention provides plasmid prACL2 comprising
Pichia ciferrii
serine palmitoyl transferase gene and a transformed
Pichia ciferrii
cell which allows an improved production of TAPS.
The inventors found that the known transformation method for
Candida utilis
(Kondo et al., J. Bacteriol., 177, 7171, 1995) can be modified and applied to the
Pichia ciferrii
. They cloned
Pichia ciferrii
ribosomal protein L41-coding gene to determine its nucleotide sequence and manipulated to give a resistance to cycloheximide, an antibiotic from yeasts, so as to be used as a selection marker. Thus recombinant gene may be linked to a plasmid which carry a desired gene to give an expression cassette which is useful to transform
Pichia ciferrii
in which the desired gene is expressed.
Moreover, the inventors succeeded in cloning of
Pichia ciferrii
GAPDH promoter gene and found that its insertion into the expression cassette allows an unexpected improvement of the expression level. In fact, they increased the production amount of TAPS by transforming the strain of
Pichia ciferrii
with the expression cassette carrying LCB2 gene as a desired gene and culturing the resulting transformed cells. LCB2 gene codes for palmitoyl transferase which is involved in the TAPS synthesis in the living body.
SUMMARY OF THE INVENTION
Accordingly, an object of the present invention is to determine and use genetic information of
Pichia ciferrii
ribosomal protein L41 gene.
Another object of the present invention is to provide an expression cassette for transforming
Pichia ciferrii
, which comprises
Pichia ciferrii
ribosomal DNA,
Pichia ciferrii
L41 gene as a selection marker, and a desired gene. In one preferred embodiment of the present invention, the marker is a gene conferring resistance to an antibiotic cycloheximide.
Another object of the present invention is to provide a method for transforming
Pichia ciferrii
with a plasmid containing the expression cassette.
The present invention determines and uses genetic information of
Pichia ciferrii
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene and GAPDH promoter gene.
The present invention provides an expression cassette for transforming
Pichia ciferrii
, which comprises
Pichia ciferrii
ribosomal DNA,
Pichia ciferrii
L41 gene as a selection marker,
Pichia ciferrii
GAPDH promoter gene and a desired gene. In one preferred embodiment of the present invention, the marker is a gene conferring resistance to an antibiotic cycloheximide.
The present invention further provides an expression cassette for transforming
Pichia ciferrii
, which comprises
Pichia ciferrii
ribosomal DNA,
Pichia ciferrii
L41 gene as a selection marker,
Pichia ciferrii
GAPDH promoter gene, a desired gene and
Pichia ciferrii
ribosomal DNA. In one preferred embodiment of the present invention, the marker is a gene conferring resistance to an antibiotic cycloheximide.
The present invention determines and uses genetic information of
Pichia ciferrii
serine palmitoyl transferase which is involved in TAPS synthesis.
The present invention provides plasmid prACL2 comprising an expression cassette having
Pichia ciferrii
serine palmitoyl transferase gene and a transformed
Pichia ciferrii
cell with an improved production of TAPS.
The present invention further provides plasmid prACGL2 comprising an expression cassette having
Pichia ciferrii
serine palmitoyl transferase gene and a
Pichia ciferrii
transformant with an improved production of TAPS.
The present invention further provides plasmid prHECGL2 comprising an expression cassette having
Pichia ciferrii
serine palmitoyl transferase gene and a
Pichia ciferrii
transformant with an improved production of TAPS.
The present invention still further provides a method for producing TAPS by culturing the transformed
Pichia ciferrii
cells.
The objects mentioned above, other features and applications of the present invention would be much more apparent by those of ordinary skills in the art from the following explanation in detail.
REFERENCES:
patent: 4851350 (1989-07-01), Stevens, Jr. et al.
patent: 5068187 (1991-11-01), Takeichi et al.
patent: 5618706 (1997-04-01), Casey et al.
patent: 5849524 (1998-12-01), Kondo et al.
patent: 0 300 287 (1989-01-01), None
patent: 0 688 871 (1995-12-01), None
patent: WO 94/10131 (1994-05-01), None
Ngo et al. Computational complexity, protein structure prediction, and the Levinthal paradox. In: The protein folding problem and tertiary structure prediction (Merz et al., eds.), Birkhauser, Boston, pp. 491-495, 1994.*
Rudinger, J. Characteristics of the amino acids as components of a peptide hormone sequence. In: Peptide hormones (Parsons, J.A., ed.), University Park Press, Baltimore, pp. 1-7, 1976.*
Yechezkel Barenholz, Nathan Godot, Eliyahu Valk, Shimon Gatt, Identification of the enzymatic lesion responsible for the accumulation of acetylated sphingosine bases in the yeastHansenula ciferr, Biochimica et Biophysica Acta, 306, pp. 341-345, (1973).
Lynfred J. Wickerham and Frank H. Stodola; Formation Of Extracellular Sphingolipides By Microorganisms; J. Bacteriol., vol. 80, pp. 484-491, (1960).
Yuzo Yamad, Kojiro Maeda, and Kozaburo Mikata: The Phylogenetic Relationships of the Hat-shaped Ascospore-forming, Nitrate-assimilatingPichiaSpecies, Formerly Classified in the GenusHansenulaSYDOW et SYDOW, Based on the Partial Sequences of 18S and 26S Ribosomal RNAs (Isaccharomycetaceae): The Proposals of Three New Genera, Ogataea, Kuraishia, and Nakazawae: Biosci; Biotech. Biochem., 58(7), pp. 1245-1257, (1994).
Keiji Kondo, Toshiko Saito, Susmu Kajiwara, Masamichi Takagi, Norihiko Misawa, A Transformation System for the YeastCandida utilis: Use of a Modified Endogenous Ribosomal Protein Gene as a Drug-Resistant Marker and Ribosomal DNA as an Integration Target for Vector DNA, Journal of Bacteriology, vol. 177, pp. 7171-7177, (1995).
Klass Nico Faber, Peter Haima, Wim Harder, Marten Veenhuis, Geert AB, Highly-efficient electrotransformation of the yeastHansenula polymorpha, Curr. Genet.
Bae Jung Hoon
Choi Eui Sung
Kang Hyun Ah
Park Chang Seo
Rhee Sang Ki
Doosan Corporation
Ketter James
Nguyen Quang
Nixon & Vanderhye
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