By a News Reporter-Staff News Editor at Drug Week — A patent application by the inventors Olsen, Carsten (Bagsvaerd, DK); Rasmussen, Michael Dolberg (Vallensbaek, DK), filed on February 14, 2014, was made available online on July 3, 2014, according to news reporting originating from Washington, D.C., by NewsRx correspondents (see also Patents).
This patent application has not been assigned to a company or institution.
The following quote was obtained by the news editors from the background information supplied by the inventors: “In the industrial production of polypeptides it is of interest to achieve a product yield as high as possible. One way to increase the yield is to increase the copy number of a gene encoding a polypeptide of interest. This can be done by placing the gene on a high copy number plasmid, however plasmids are unstable and are often lost from the host cells if there is no selective pressure during the cultivation of the host cells. Another way to increase the copy number of the gene of interest is to integrate it into the host cell chromosome in multiple copies.
“The present day public debate concerning the industrial use of recombinant DNA technology has raised some questions and concerns about the use of antibiotic resistance marker genes. Antibiotic marker genes are traditionally used as a means to select for strains carrying multiple copies of both the marker genes and an accompanying expression cassette coding for a polypeptide of industrial interest. In order to comply with the current demand for recombinant production host strains devoid of antibiotic markers, we have looked for possible alternatives to the present technology that will allow substitution of the antibiotic markers we use today with non-antibiotic marker genes.
“WO 02/00907 (Novozymes, Denmark) discloses a method for stable chromosomal multi-copy integration of genes into a production host cell in specific well-defined sites. It is disclosed to first render a recipient cell deficient by inactivating one or more conditionally essential gene, e.g., to make the cell auxotrophic for an amino acid. A gene of interest may then be integrated into the chromosome along with a DNA sequence which complements the deficiency of the cell, thus making the resulting cell selectable; the Bacillus licheniformis metC gene is disclosed as a conditionally essential marker herein.
“WO 01/90393 (Novozymes, Denmark) discloses a method for increasing the gene copy number in a host cell by gene-amplification, without leaving antibiotic resistance markers behind in the host cell. The disclosed method relies on rendering a specific type of conditionally essential chromosomal gene of the host cell non-functional. A single amplification unit comprising the gene of interest, and a DNA sequence, which when integrated into the chromosome complements the non-functional conditional essential chromosomal gene, is integrated into the chromosome.
“In order to provide recombinant production strains devoid of antibiotic resistance markers, it remains of industrial interest to find new methods to stably integrate genes in multiple copies into host cell chromosomes. Even incremental improvements of existing methods or mere alternatives are of considerable interest to the industry.”
In addition to the background information obtained for this patent application, NewsRx journalists also obtained the inventors’ summary information for this patent application: “The problem to be solved by the present invention is to provide alternative host cells comprising multiple copies of a gene of interest, which cells are devoid of antibiotic markers, for use in the industrial production of polypeptides in high yields.
“The solution is based on the observation that an amplification unit can be integrated into the chromosome of a host cell, and subsequently be amplified, without the use of classical antibiotic markers, antibiotics, or endogenously produced inhibitory compounds.
“In traditional amplification protocols, higher gene expression is a result of duplications of the antibiotic resistance marker gene, duplications which are selected in stepwise cultivation and selection rounds by adding increasing amounts of the antibiotic compound to the cultivation medium in each cultivation step.
“A cell which has become auxotrophic, e.g., due to a non-functional conditionally essential gene, would normally be complemented back to the prototrophic phenotype by the integration (or restoration) in the chromosome of even one single functional copy of the non-functional gene. Since normally only one copy is needed, such genes have not previously been attractive candidates for amplification purposes.
“However, the present inventors lowered the expression-level of a non-antibiotic conditionally essential gene by decreasing the promoter activity, so that more than one functional copy of the gene would be advantageous to a deficient host cell. The integration of an amplification unit comprising such a low-level expression conditionally essential gene, into a host cell deficient for the same gene, reproducibly resulted in genomic duplications of the integrated amplification unit, comparable to what has been observed when using traditional amplifiable antibiotic markers.
“In fact, this invention provides the means for controlling the level of gene expression, i.e., copy-number, in a host cell. By choosing carefully the strength of the heterologous promoter expressing the conditionally essential marker gene in the amplification unit, the optimal copy-number of the amplification unit can be adjusted up or down, depending on the desired expression level of the gene of interest also comprised in the unit.
“Accordingly, in a first aspect the invention relates to a bacterial host cell comprising at least two copies of an amplification unit in its genome, said amplification unit comprising: i) at least one copy of a gene of interest, and ii) an expressible conditionally essential gene, wherein the conditionally essential gene is either promoterless or transcribed from a heterologous promoter having an activity substantially lower than the endogenous promoter of said conditionally essential gene, and wherein the conditionally essential gene if not functional would render the cell auxotrophic for at least one specific substance or unable to utilize one or more specific sole carbon source.
“In a second aspect, the invention relates to a method for producing a protein encoded by a gene of interest, comprising a) culturing a bacterial host cell comprising at least two duplicated copies of an amplification unit in its genome, the amplification unit comprising: i) at least one copy of the gene of interest, and ii) an expressible conditionally essential gene, wherein the conditionally essential gene is either promoterless or transcribed from a heterologous promoter having an activity substantially lower than the endogenous promoter of said conditionally essential gene, wherein the conditionally essential gene if not functional would render the cell auxotrophic for at least one specific substance or unable to utilize one or more specific sole carbon source; and b) recovering the protein.
“In a final aspect, the invention also relates to a method for producing a bacterial cell comprising two or more amplified chromosomal copies of a gene of interest, the method comprising: a) providing a bacterial cell comprising at least one copy of an amplification unit, the unit comprising: i) at least one copy of the gene of interest, and ii) an expressible functional copy of a conditionally essential gene, which is either promoterless or transcribed from a heterologous promoter having an activity substantially lower than the endogenous promoter of said conditionally essential gene, wherein the conditionally essential gene if not functional would render the cell auxotrophic for at least one specific substance or unable to utilize one or more specific sole carbon source; b) cultivating the cell under conditions suitable for growth in a medium deficient of said at least one specific substance and/or with said one or more specific sole carbon source, thereby providing a growth advantage to a cell in which the amplification unit has been duplicated in the chromosome; and c) selecting a cell wherein the amplification unit has been duplicated in the chromosome, whereby two or more amplified chromosomal copies of the gene of interest were produced.
“It is envisioned that all the preferred embodiments of the cell of the invention that are shown herein would be suitable for use in the methods of the second and third aspects of the invention”
URL and more information on this patent application, see: Olsen, Carsten; Rasmussen, Michael Dolberg. Method for Stable Gene-Amplification in a Bacterial Host Cell. Filed February 14, 2014 and posted July 3, 2014. Patent URL: http://appft.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&Sect2=HITOFF&u=\%2Fnetahtml\%2FPTO\%2Fsearch-adv.html&r=3108&p=63&f=G&l=50&d=PG01&S1=20140626.PD.&OS=PD/20140626&RS=PD/20140626
Keywords for this news article include: Patents, Technology, Chromosomes, Cell Nucleus, Intranuclear Space, Cellular Structures, Intracellular Space.
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