Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-11-19
2001-12-11
Ketter, James (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S069100, C435S483000, C435S200000, C435S254200, C435S320100, C536S023100, C536S024100, C536S024300, C536S024500
Reexamination Certificate
active
06329141
ABSTRACT:
TECHNICAL FIELD
The present invention relates to methods for transforming Phaffia yeast, transformed Phaffia strains, as well as recombinant DNA for use therein.
BACKGROUND OF THE INVENTION
Methods for transforming the yeast
Phaffia rhodozyma
have been disclosed in European patent application 0 590 707 A1. These methods involve incubation of protoplasts with DNA or incubation of Phaffia cells with DNA followed by lithium acetate treatment. The recombinant DNA used to transform Phaffia strains with either of these methods comprised a Phaffia actin gene promoter to drive expression of the selectable marker genes coding for resistance against G418 or phleomycin. The methods involve long PEG and lithium acetate incubation times and transformation frequencies are low. When protoplasts are used, the transformation frequency is dependent on the quality of the protoplast suspension, making the procedure less reliable.
Recently a method for transforming Phaffia strains has been reported by Adrio J. L. and Veiga M. (July 1995, Biotechnology Techniques Vol. 9, No. 7, pp. 509-512). With this method the w transformation frequencies are in the range of 3 to 13 transformants per &mgr;g DNA, which is low. A further disadvantage of the method disclosed by these authors consists in increased doubling time of the transformed cells. The authors hypothesised that this may be due to interference of the autonomously replicating vector with chromosome replication.
Clearly, there is still a need for a reliable and efficient method of transforming Phaffia strains with foreign DNA. It is an objective of the present invention to provide methods and means to achieve this. It is a further objective of the invention to optimize expression of certain genes in
Phaffia rhodozyma
in order to make Phaffia a more suitable production host for certain valuable compounds.
SUMMARY OF THE INVENTION
The invention provides a method for obtaining a transformed Phaffia strain, comprising the steps of contacting cells or protoplasts of a Phaffia strain with recombinant DNA under conditions conducive to uptake thereof, said recombinant DNA comprising a transcription promoter and a downstream sequence to be expressed which is heterologous to said transcription promoter, in operable linkage therewith, identifying
Phaffia rhodozyma
cells or protoplasts having obtained the said recombinant DNA in expressible form, wherein the transcription promoter comprises a region that is found upstream of the open reading frame of a highly expressed Phaffia gene. According to a preferred embodiment of the invention said highly expressed Phaffia gene is a glycolytic pathway gene, more preferably the glycolytic pathway gene is coding for Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH). According to one aspect of the invention, said heterologous downstream sequence comprises an open reading frame coding for resistance against a selective agent, such as G418 or phleomycin.
Another preferred method according to the invention is one, wherein said recombinant DNA comprises further a transcription terminator downstream from the said DNA to be expressed, in operable linkage therewith, which transcription terminator comprises a region found downstream of the open reading frame of a Phaffia gene. It is still further preferred, that the recombinant DNA is in the form of linear DNA.
Another preferred embodiment comprises, in addition to the steps above, the step of providing an electropulse after contacting of Phaffia cells or protoplasts with DNA.
According to another embodiment the invention provides a transformed Phaffia strain capable of high-level expression of a heterologous DNA sequence, which strain is obtainable by a method according to the invention. Preferably, said Phaffia strain contains at least 10 copies of the said recombinant DNA integrated into its genome, such as a chromosome, particularly in the ribosomal DNA locus of said chromosome.
The invention also provides recombinant DNA comprising a transcription promoter and a heterologous downstream sequence to be expressed, in operable linkage therewith, wherein the transcription promoter comprises a region found upstream of the open reading frame of a highly expressed Phyla gene, preferably a glycolytic pathway gene, more preferably a gene coding for Glyceraldehyde-3-Phosphate Dehydrogenase.
Also provided is recombinant DNA according to the invention, wherein the heterologous downstream sequence comprises an open reading frame coding for reduced sensitivity against a selective agent, preferably G418 or phleomycin. Said recombinant DNA preferably comprises further a transcription terminator downstream from the said heterologous DNA sequence to be expressed, in operable linkage therewith.
Further aspects of the invention concern a microorganism harbouring recombinant DNA according to the invention, preferably Phaffia strains, more preferably
Phaffia rhodozyma
strains, as well as cultures thereof.
According to still other preferred embodiments isolated DNA fragments are provided comprising a Phaffia GAPDH-gene, or a fragment thereof, as well as the use of such a fragment for making a recombinant DNA construct. According to one embodiment of this aspect said fragment is a regulatory region located upstream or downstream of the open reading frame coding for GAPDH, and it is used in conjunction with a heterologous sequence to be expressed under the control thereof
The invention according to yet another aspect, provides a method for producing a protein or a pigment by culturing a Phaffia strain under conditions conducive to the production of said protein or pigment, wherein the Phaffia strain is a transformed Phaffia strain according to the invention.
According to another aspect of the invention, a method for obtaining a transformed Phaffia strain, comprising the steps of
contacting cells or protoplasts of a Phaffia strain with recombinant DNA under conditions conducive to uptake thereof,
said recombinant DNA comprising a transcription promoter and a downstream sequence to be expressed in operable linkage therewith,
identifying
Phaffia rhodozyma
cells or protoplasts having obtained the said recombinant DNA in expressible form,
wherein the downstream am sequence to be expressed comprise s an isolate d DNA sequence coding for an enzyme involved in the carotenoid biosynthetic pathway of
Phaffia rhodozyma.
Preferably, said enzyme has an activity selected from geranylgeranyl pyrophosphate synthase (crtE), phytoene synthase (crtB), phytoene desaturase (crtI) and lycopene cyclase (crtY), more preferably an enzyme having an amino acid sequence selected from the one represented by SEQIDNO: 13, SEQIDNO: 15, SEQIDNO: 17 and SEQIDNO: 19. According to a further embodiment, the transcription promoter is heterologous to said isolated DNA sequence, such as a glycolytic pathway gene in Phaffia. Especially preferred according to this embodiment is the Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) gene promoter.
Also provided is a transformed Phaffia strain obtainable by a method according to the invention and capable of expressing preferably over-expressing the DNA sequence encoding an enzyme, involved in the carotenoid biosynthesis pathway gene.
The invention is also embodied in recombinant DNA comprising an isolated DNA sequence according to the invention, preferably in the form of a vector.
Also claimed is the use of such a vector to transform a host, such as a Phaffia strain:
A host obtainable by transformation, optionally of an ancestor, using a method according to any one of claims
1
to
5
, wherein said host is preferably capable of over-expressing DNA according to the invention.
According to a further embodiment a method is provided for expressing an enzyme involved in the carotenoid biosynthesis pathway, by culturing a host according to the invention under conditions conducive to the production of said enzyme. Also provided is a method for producing a carotenoid by cultivating a host according to the invention under conditions conducive to the production of carotenoid.
The following fi
Van Ooijen Albert Johannes Joseph
Verdoes Jan Cornelis
Wery Jan
DSM N.V.
Ketter James
Morrison & Foerster / LLP
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