Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-10-25
2004-04-20
Myers, Carla J. (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S029000, C435S091100, C435S091410, C435S091500
Reexamination Certificate
active
06723504
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a method of generating a gene library from an environmental pool of organisms, which gene library is enriched in DNA encoding a polypeptide with an activity of interest.
BACKGROUND OF THE INVENTION
The advent of recombinant DNA techniques has made it possible to select single protein components with interesting properties and produce them on a large scale. This represents an improvement over the previously employed production process using microorganisms isolated from nature and producing a mixture of proteins which would either be used as such or separated after the production step. Methods have been developed for rapid identification of genes encoding a polypeptide of interest.
One example is the so called expression cloning technique described in WO 93/11249 (Novo Nordisk A/S). The technique disclosed in WO 93/11249 comprises a method of screening for a DNA sequence in a DNA library prepared from an organism suspected of producing genes encoding polypeptides with activities of interest. Such a library has traditionally been made on DNA isolated from a single known microorganism.
A compartmentalization method of screening microorganisms having a selectable characteristic has previously been devised in WO 97/37036, and a process for forming a normalized genomic DNA library from an environmental sample is described in WO 97/37036.
However, a method of generating a gene library from an environmental pool of organisms, which gene library is enriched in DNA encoding a polypeptide with an activity of interest has never been described. Therefore, it would be desirable to have a method based on biological enrichment for selecting potentially interesting genes from environmental pool of organisms.
SUMMARY OF THE INVENTION
It has now been found possible to use biological enrichment for selecting potentially interesting genes from an environmental pool of organisms. Accordingly, the invention provides a method for generating a gene library from an environmental pool of organisms, which gene library is enriched in DNA encoding a polypeptide with an activity of interest, which method comprises:
a) subjecting the environmental pool of organisms to cultivation in a medium and/or under conditions suitable for enriching said pool of organisms in organisms harbouring said DNA; and
b) preparing a gene library from the resulting enriched pool of organisms.
The invention also provides a method of selecting a DNA sequence of interest from an environmental pool of organisms, which method comprises:
a) subjecting the environmental pool of organisms to cultivation in a medium and/or conditions suitable for enriching said pool of organisms in organisms harbouring said DNA sequence;
b) producing gene libraries from the resulting enriched pool of organisms;
c) screening the libraries for DNA containing the desired gene; and
d) selecting the DNA sequence of interest resulting from the screening of step c).
Further, the invention relates to a gene library prepared from an enriched environmental pool of organisms enriched in DNA encoding a polypeptide with an activity of interest.
DETAILED DISCLOSURE OF THE INVENTION
It is an object of the present invention to provide a method for generating a gene library from an environmental pool of organisms, which gene library is enriched in DNA encoding a polypeptide with an activity of interest, which method comprises:
a) subjecting the environmental pool of organisms to cultivation in a medium and/or under conditions suitable for enriching said pool of organisms in organisms harbouring said DNA; and
b) preparing a gene library from the resulting enriched pool of organisms.
In the context of the present invention, the term “an environmental pool of organisms” means a environmental sample comprising microorganisms and cells from higher animals harboring DNA encoding a polypeptide with an activity of interest. The environmental sample may for instance be an environmental sample of soil or plant material, animal or insect dung, insect gut, animal stomach, a marine sample of sea or lake water, sewage, waste water, a sample of sludge or sediment, etc., comprising one or, as in most case, a vast number of different microorganisms or living cells.
In step a), the sample as such is cultivated without any need for further purification. By selecting the medium and the cultivation conditions at which the sample is cultivated, it is possible for enriching or (amplifying) organisms having optimal growth at the specific cultivation conditions, and expressing polypeptides with properties adapted to the cultivation conditions. The gene library prepared in step b) may be prepared by any suitable technique known in the art, non-limiting examples of which are described in Example 3 and 4.
The advantage presented by the present screening method is primarily that the rate at which novel genes may be isolated and, consequently, novel products be developed may be greatly increased. Furthermore, the method permits screening for multiple polypeptides activities and may even result in the isolation of several different genes coding for the same type of polypeptides.
By use of the invention it is possible to exploit enriched cultures for detecting novel enzymes, and other polypeptides with an activity of interest.
In a preferred embodiment, the method of the invention comprises subjection the environmental pool of organisms to cultivation in a medium, which contains a substrate for the polypeptide with the desired activity. A wide range of substrates for the enrichment of the environmental of organisms containing different types of gene products may be used. For instance, a DNA encoding a polypeptide with an activity of interest such as a pectinase enzyme may be selected as a gene product on a substrate as pectin.
In a preferred embodiment, the substrate constitutes the carbon source and/or nitrogen source of the medium.
In a more preferred embodiment, the substrate comprises pectin, amylose, cellulose, galactan, xylan, arabinan, mannan, lipid or hemicellulose or a combination thereof.
In a preferred embodiment of the method of the invention, the enrichment is achieved by one or more growth conditions. In a another preferred embodiment, the growth conditions comprise pH and temperature. In yet another preferred embodiment, the growth conditions of step a) used for achieving the enrichment comprises any pH range ie. 0-12, preferably of about 6-9, in particular 9-12, at any temperature range i.e. 0-120° C., preferably about 25-30°, preferably 30-500°, most preferred 50-70° C.
An important step in the procedure for selection of a potentially interesting environmental pool of organisms is to select the optimal pool to start from. To select genes encoding polypeptides that can break down natural compounds of plant (or animal) origin, it is preferable to look into those biotopes in nature where such materials are efficiently decomposed. Examples of animals especially efficient in breaking down plant material are the ruminates, termites and insects (sensu lato).
In a preferred embodiment, the environmental pool of microorganisms is isolated from an animal stomach or an insect gut.
In a more preferred embodiment, the pool of microorganisms is isolated from a cow's rumen.
Likewise, it is important when selecting genes encoding polypeptides with an activity of interest that are capable of working under e.g. strongly alkaline conditions, to isolate the pool of organisms from an equally strongly alkaline biotope. It is known in the art that in order for
Bacillus thuringensis
(
Bt
) toxins to be active, strongly alkaline conditions are a prerequisite [
Bacillus thuringensis
, an environmental biopesticide: Theory and Practice, 1993, eds. P. F. Entwistl et al., Wiley & Son, UK]. The guts of the larvae belonging to the orders of insects known to be sensitive to the
Bt
toxins comprise environmental pools of a high alkaline nature (approx pH 10). Such insect orders are especially Isoptera, Lepidoptera, Coleoptera and Diptera.
Consequently i
Duffner Fiona
Lange Lene
Sandal Thomas
Schäfer Thomas
Sjøholm Carsten
Garbell Jason
Johannsen Diana B.
Lambiris Elias
Myers Carla J.
Novozymes A/S
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