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
2001-07-05
2004-07-13
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...
C435S041000, C435S071100, C435S071200, C435S440000, C435S471000, C435S476000, C435S477000, C435S479000, C435S481000, C435S485000, C435S243000, C435S252100, C435S252300, C435S252310, C435S252500, C435S320100
Reexamination Certificate
active
06762040
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to a method for increasing the copy number of a chromosomally integrated expression cassette in a microbial strain without leaving antibiotic resistance markers behind in the strain, the necessary genetic constructs, and the strains used in and resulting from the method of the invention. It is desirable for the biotech industry to provide microbial strains devoid of antibiotic resistance markers comprising several chromosomally integrated copies of a gene of interest, for the industrial high yield production of polypeptides.
BACKGROUND OF THE INVENTION
The present debate concerning the industrial use of recombinant DNA technology has raised some questions and concern about the use of antibiotic marker genes. An antibiotic marker gene is traditionally used as a means to select for strains carrying multiple copies of both the marker gene and an accompanying expression cassette coding for a polypeptide of industrial interest. Amplification of the expression cassette by increasing the copy number in a microbiological production strain is desirable because there is very often a direct correlation between the number of copies and the final product yields. The amplification method using antibiotic selection has been used extensively in many host strains over the past 15 years and has proven to be a very efficient way to develop high yielding production strains in a relatively short time, irrespective of the expression level of the individual expression cassettes.
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 new marker genes.
The catabolic pathway of galactose in bacilli is very similar to the pathway of other sugars. The carbon molecule is transported into the cell via a permease, a kinase charges the molecule with a phosphate group and a transferase reaction transfers the phosphate group to a glucose molecule which is then shuttled directly into the glycolytic pathway. In the case of galactose catabolism the transferase reaction generates UDP-galactose as a sideproduct which is a very toxic compound for all living cells. This compound is normally converted to UDP-glucose by an epimerase coded for by the galE gene. The use of galE in a simple selection method for plasmid transformed cells, especially plant cells, is mentioned in WO 00/09705.
SUMMARY OF THE INVENTION
The problem to be solved by the present invention is to increase the copy number of a chromosomally integrated expression cassette in a microbial strain in a way by which a resulting host cell devoid of antibiotic markers is provided for the use in industrial production of polypeptides in high yields.
The solution is based on that the present inventors demonstrated that a nucleotide construct comprising an amplification unit as defined herein can integrate into the chromosome of a host cell and increase in number of chromosomally integrated copies without the use of classical antibiotic markers or antibiotics.
Accordingly, in a first aspect the invention relates to a method for increasing the number of copies of an amplification unit integrated into a host cell chromosome, wherein the method comprises the steps of:
a) rendering a chromosomal gene of a host cell non-functional, wherein the host cell becomes susceptible to an inhibitory compound endogenously produced by the host cell when the host cell is cultivated in a medium comprising a precursor;
b) making a nucleic acid construct comprising an amplification unit, wherein the unit comprises:
i) an expression cassette comprising at least one copy of a gene of interest; and
ii) an expressable copy of the chromosomal gene of step a), wherein the unit integrates into the host cell chromosome;
c) introducing the nucleic acid construct of step b) into the host cell of step a), wherein at least one copy of the amplification unit integrates into the host cell chromosome;
d) cultivating the host cell of step c) in a medium comprising the precursor, wherein a chromosomally integrated copy of the amplification unit is duplicated or multiplied on the host cell chromosome;
e) selecting a host cell comprising two or more chromosomally integrated copies of the amplification unit; and optionally
f) performing one or more cycles of steps d) and e) using the host cell selected in step e) in each new cycle; wherein the number of chromosomally integrated copies of the amplification unit increases with each repeat.
Further, in a second aspect the invention relates to a method for constructing a host cell comprising at least one copy of an amplification unit integrated into the host cell chromosome, wherein the method comprises the steps of:
a) rendering a chromosomal gene of a host cell non-functional, wherein the host cell becomes susceptible to an inhibitory compound endogenously produced by the host cell when the host cell is cultivated in a medium comprising a precursor;
b) making a nucleic acid construct comprising an amplification unit, wherein the unit comprises:
i) an expression cassette comprising at least one copy of a gene of interest; and
ii) an expressable copy of the chromosomal gene of step a), wherein the unit integrates into the host cell chromosome;
c) introducing the nucleic acid construct of step b) into the host cell of step a) and cultivating the host cell in a medium comprising the precursor, wherein at least one copy of the amplification unit integrates into the host cell chromosome; and
d) selecting a host cell comprising at least one chromosomally integrated copy of the amplification unit.
A third aspect of the invention relates to a method for increasing the number of copies of an amplification unit integrated into a host cell chromosome, wherein the method comprises the steps of:
a) providing a host cell, wherein a chromosomal gene has been rendered non-functional, whereby the host cell becomes susceptible to an inhibitory compound endogenously produced by the host cell when the host cell is cultivated in a medium comprising a precursor;
b) introducing a nucleic acid construct into the host cell of step a), the nucleic acid construct comprising an amplification unit, wherein the unit comprises:
i) an expression cassette comprising at least one copy of a gene of interest; and
ii) an expressable copy of the chromosomal gene of step a), wherein at least one copy of the amplification unit integrates into the host cell chromosome;
c) cultivating the host cell of step b) in a medium comprising the precursor, wherein a chromosomally integrated copy of the amplification unit is duplicated or multiplied on the host cell chromosome;
d) selecting a host cell comprising two or more chromosomally integrated copies of the amplification unit; and optionally
e) performing one or more cycles of steps c) and d) using the host cell selected in step d) in each new cycle; wherein the number of chromosomally integrated copies of the amplification unit increases with each cycle.
As clear from above, genetic tools are provided for performing the method of the invention as described herein.
Accordingly in a fourth aspect the invention relates to an amplification unit comprising:
a) an expression cassette comprising at least one copy of a gene of interest; and
b) an expressable copy of a conditionally essential chromosomal gene of a host cell; wherein the unit integrates into the host cell chromosome upon introduction of the nucleic acid construct into the host cell.
Further in a fifth aspect the invention relates to a nucleic acid construct comprising a unit as defined in any of the previous aspects.
The method of the invention achieves the construction of a host cell comprising at least one chromosomally integrated copy of the amplification unit as defined above, where such a host cell is highly desirable for industrial production of polypeptides in high yields.
Consequently in a sixth aspect the invention relates to a host cell wherein
Ketter James
Lambertson David
Lambiris Elias
Novozymes A/S
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