Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
2000-07-11
2003-07-29
Ketter, James (Department: 1636)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S006120, C435S252300, C435S254110
Reexamination Certificate
active
06599745
ABSTRACT:
BACKGROUND OF THE INVENTION
The subject invention lies in the field of microorganism mutation and selection of the mutants. In particular the invention is directed at obtaining metabolic mutants in a simple, direct and specific manner. In a preferred embodiment it is also possible to obtain desired mutants not comprising recombinant DNA, thereby facilitating incorporation thereof in products for human consumption or application, due to shorter legislative procedures. The method according to the invention involves random mutation and specific selection of the desired metabolic mutant. The method can suitably be carried out automatically. Such a mutant can exhibit either increased or decreased metabolic activity. The specificity of the method lies in the selection conditions applied. The mutants obtained are mutated in their regulatory function with regard to a predetermined part of metabolism. Dependent on the selection conditions derepressed mutants can be isolated that will thus exhibit overexpression or mutants can be found in which particular metabolic enzymic activity is eliminated. It thus becomes possible to eliminate undesirable metabolic enzymic activity or to increase desirable metabolic activity.
The methods according to the invention can suitably be carried out on well characterised sources that are already widely used in industry. The overexpressing mutants can for example be used as major sources of enzymes producing huge amounts at low cost. The initial strain to be mutated will depend on several factors known to a person skilled in the art such as: efficient secretion of proteins, the availability of large scale production processes, experience with downstream processing of fermentation broth, extensive genetic knowledge and the assurance of using safe organisms.
In another aspect of the invention it has now also become possible to ascertain and identify specific metabolic gene regulating functions.
To date a method for preparing mutants that was industrially applicable and could be automated was a method of mutating without selection and subsequent analysis of the mutants for the aspect which was to be amended. An alternative method with selection always required an enrichment step, followed by selection on the basis of growth or non growth. This meant a large number of undesired mutants had first to be weeded out. Also the existing method resulted in a high number of mutants with an incorrect phenotype and thus exhibits low selectivity.
Some years ago Gist-Brocades developed and introduced the pluGBug marker gene free technology for
Aspergillus niger
. In the GIST 94/60 p 5-7 by G. Selten a description is given of a vector for
Aspergillus niger
comprising glucoamylase regulatory regions to achieve high expression levels of the gene it regulates. This was selected as regulatory region on the basis of the naturally high expression of glucoamylase by native
Aspergillus niger
. Using recombinant DNA techniques the regulatory region was fused appropriately to the gene of interest as was the selection marker Aspergillus AmdS, allowing selection of the desired transformants after transferring the expression cassette to the
A. niger
host. Multiple copies of the expression cassette then become randomly integrated into the
A. niger
genome. The enzyme produced as described in the article was phytase. Subsequently the generation of marker free transformants can be achieved. In the known system the generation of marker free recombinant strains is actually a two step process since the amdS gene can be used bidirectionally. First in a transformation round to select initial transformants possessing the offered expression cassette and subsequently in a second round by counterselecting for the final recombinant strain which has lost the amdS gene again. The amdS gene encodes an enzyme which is able to convert acetamide into ammonium and acetate. Acetamide is used as sole N-source in the transformation round. In the recombination round fluoro acetamide is used as selective N-source, with a second appropriate N source such as e.g. urea. As the product fluoroacetate is toxic for other cells the propagation will be limited to those cells which have lost the amdS gene by an internal recombination event over the DNA repeats within the expression cassette. The largest problem with the known method is the fact that the resulting strain is a recombinant strain. The desired characteristic has to be introduced by incorporation of “foreign” nucleic acid, which can lengthen the time required for and sometimes even prevent legislative approval. In addition the method is not suited for developing strains with amended metabolism. Due to the presence of enzyme cascades and multiple feedback loops the mere incorporation of a particular gene cannot always lead to the desired result. Overproduction of a particular product as encoded can be compensated for by concomitant overexpression of another product or down regulated thus annulling the effect of the incorporated gene. The incorporation of DNA will therefore often be a case of trial and error with the incorporation of the desired nucleic acid being selectable but the desired phenotype not necessarily concomitantly being achieved. Furthermore the loss of the marker gene is a spontaneous process which takes time and cannot be guaranteed to occur for all transformants comprising the nucleic acid cassette.
It is known that strain improvement in microorganisms can be achieved by modification of the organism at different levels. Improvement of gene expression at the level of transcription is mostly achieved by the use of a strong promoter, giving rise to a high level of mRNA encoding the product of interest, in combination with an increase of gene dosage of the expression cassette. Although this can lead to an increase of the product formed, this strategy can have a disadvantage in principle. Due to the presence of multiple copies of the promoter the amount of transcriptional regulator driving transcription can become limited, resulting in a reduced expression of the target gene or genes of the regulator. This has been observed in the case of
Aspergillus nidulans
strains carrying a large number of copies of the amdS gene (Kelly and Hynes, 1987; Andrianopoulos and Hynes, 1988) and in the case of
A. nidulans
strains carrying multiple copies of a heterologous gene under the alcA promoter (Gwynne et al., 1987). In the latter case an increase of the alcR gene, encoding the transcriptional regulator of the alcA gene, resulted in the increase of expression of the expression cassette (Gwynne et al., 1987; Davies, 1991). In analogy to the effects found in
Aspergillus nidulans
, in
Aspergillus niger
similar limitations were observed in using the glucoamylase (glaA) promoter, due to limitation of the transcriptional regulator driving transcription (Verdoes et al. 1993; Verdoes et al 1995; Verdoes, 1994). Cloning of the glaA regulatory gene has thusfar been hampered by lack of selection strategy.
In the case of the arabinase gene expression a clear competition for transcriptional regulator was found upon the increase of arabinase gene dosage (Flipphi et al. 1994), reflecting a limitation of a transcriptional regulator common to all three genes studied.
In addition to the abovementioned drawbacks of the state of the art isolation and determination of regulator genes has until now been extremely difficult due to the fact that most of the regulatory proteins exist in very low concentrations in the cell making it difficult to determine which substance is responsible for regulation. In addition generally the regulatory product is not an enzyme and can only be screened for by a DNA binding assay which makes it difficult to determine and isolate and is very time consuming. Thus far the strategies used to clone regulatory genes are e.g.:
by complementation, which however requires a mutant to be available,
by purification of the regulatory protein, which is extremely laborious, since the protein can only be characterised by its DNA binding properties. Som
De Graaff Leendert Hendrik
Van Den Broeck Henriëtta Catharina
Visser Jacob
Danisco Ingredients A/S (Danisco A/S)
Ketter James
Loeb Bronwen M.
Sughrue & Mion, PLLC
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