EP1124948A1 - Procede de creation d'une banque de genes - Google Patents

Procede de creation d'une banque de genes

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Publication number
EP1124948A1
EP1124948A1 EP99948722A EP99948722A EP1124948A1 EP 1124948 A1 EP1124948 A1 EP 1124948A1 EP 99948722 A EP99948722 A EP 99948722A EP 99948722 A EP99948722 A EP 99948722A EP 1124948 A1 EP1124948 A1 EP 1124948A1
Authority
EP
European Patent Office
Prior art keywords
organisms
pool
interest
dna
enzyme
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP99948722A
Other languages
German (de)
English (en)
Inventor
Thomas Sandal
Carsten Sjoholm
Thomas Schaefer
Lene Lange
Fiona Duffner
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Novozymes AS
Original Assignee
Novozymes AS
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Novozymes AS filed Critical Novozymes AS
Publication of EP1124948A1 publication Critical patent/EP1124948A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1093—General methods of preparing gene libraries, not provided for in other subgroups

Definitions

  • 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.
  • WO 93/11249 Novartis 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.
  • 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 :
  • step c) 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) .
  • 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.
  • 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.
  • step a) the sample as such is cultivated without any need for further purification.
  • 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.
  • 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 substrate for the polypeptide with the desired activity may be used.
  • 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.
  • the substrate constitutes the carbon source and/or nitrogen source of the medium.
  • the substrate comprises pectin, amylose, cellulose, galactan, xylan, arabinan, mannan, lipid or hemicellulose or a combination thereof.
  • the enrichment is achieved by one or more growth conditions.
  • the growth conditions comprise pH and temperature.
  • the growth conditions of step a) used for achieving the enrichment comprises any pH range i.e. 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 30-50°, most preferred 50- 70°C.
  • the environmental pool of microorganisms is isolated from an animal stomach or an insect gut .
  • the pool of microorganisms is isolated from a cow's rumen.
  • 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 in a preferred embodiment, the pool of microorganisms is isolated from the gut of an insect of the Isoptera, Lepidoptera, Coleoptera, or Diptera families.
  • the pool of microorganisms is isolated from the gut of insects selected from the group consisting of Agrotis, Neotermes castaneus, Tineola bisselliella, and Melolontha vulgaris .
  • the pool of microorganisms is enriched by supplying feed to the animal or insect, which comprises a substrate for the polypeptide with an activity of interest .
  • DNA encoding a polypeptide with an activity of interest include among others enzymatic activity and anti-microbial activity.
  • the gene libraries are enriched in DNA encoding an enzyme activity of interest.
  • the activity of interest is an enzymatic activity, such as an activity selected from the group comprising of phosphatases oxidoreductases (E.C. 1), transferases (E.C. 2); hydrolases
  • E.C. 3 such as esterases (E.C. 3.1), in particular upases and phytase; such as glucosidases (E.C. 3.2), in particular xylanase, cellulases, hemicellulases, and amylase, such as peptidases (E.C. 3.4), in particular proteases; lyases (E.C. 4) ; isomerases (E.C. 5) ; ligases (E.C. 6) .
  • esterases E.C. 3.1
  • upases and phytase such as glucosidases (E.C. 3.2), in particular xylanase, cellulases, hemicellulases, and amylase, such as peptidases (E.C. 3.4), in particular proteases; lyases (E.C. 4) ; isomerases (E.C. 5) ; ligases (
  • the enzyme of interest comprises a protease, lipase, beta-galactosidase, lactase, polygalacturonase, beta-glucoamylase, esterase, hemicellulase, peroxidase, oxidase, laccase or glucose oxidase .
  • the enzymes obtained in the method is an amylase, in particular an ⁇ -amylase or a ⁇ -amylase, an arabinanase, an arabinofuranosidase, a galactanase, an ⁇ - galactosidase, a ⁇ -galactosidase, a polygalacturonase, a pectin methyl esterase, a rhamnogalacturonase, a rhamnogalacturon acetyl esterase, a pectin lyase, a xylanase, a cellulase, a ⁇ - glucosidase, a cellobiohydrolase, a xylosidase, a mannanase, and/or a glucuronisidase.
  • an amylase in particular an ⁇ -amylase or a ⁇ -amylase
  • the environmental pool of organisms containing DNA encoding a polypeptide with an activity of interest are typically microorganisms such as Eubacteria, Archaebacteria, fungi, algae and/or protozoa.
  • the polypeptide may be an enzyme of interest obtained from any known organism.
  • the enzyme may be obtained from microorganism, in particular from a bacteria, from a filamentous fungus or a yeast .
  • the organisms are enriched cultures meaning that the cultures are selected on a specific substrate from which other organisms are not able to grow or having a reduced growth.
  • Step a) and b) are as described above.
  • step c) clones found to comprise a DNA sequence originated from the prepared gene library in step b) may be screened for any activity of interest. Examples of such activities include enzymatic activity, anti-microbial activity or biological activities.
  • step c) gene libraries are screened for genomic DNA containing the desired gene, and in step d) , the DNA sequence of interest are selected from the screening of step c) .
  • Step c) and d) may be performed by standard methods known in the art .
  • polypeptide with the activity of interest may then be tested for a desired performance under specific conditions and/or in combination with e . g. chemical compounds or agents.
  • the gene libraries may be screened according to the method of the invention for a polypeptide with an activity of interest e.g. a specific activity, and/or a specific property of interest such as thermostability, high pH tolerance, wash performance, textile dyeing, hair dyeing or bleaching properties, a effect in feed or food ect .
  • an activity of interest e.g. a specific activity, and/or a specific property of interest such as thermostability, high pH tolerance, wash performance, textile dyeing, hair dyeing or bleaching properties, a effect in feed or food ect .
  • the appropriate assay for testing for a desired activity and/or property well be known to the skilled person.
  • the gene library comprises an enzyme-encoding gene of interest, and the gene library is screened for enzymes under conditions which the enzyme is active.
  • the library may be screened for enzymes at e.g. high temperatures such as 60-110°C and high pH such as 10-12 e.g. in cases where it is desired to isolate a DNA sequence encoding an alkaline enzyme with a relatively high thermostability.
  • pH can be in any range e.g. of from about 0 to about 12, and the temperature in any range e.g. of from about 5 to about 110°C, preferably of from about 60 to about 90°C.
  • the gene library comprises a polypeptide with an activity of an enzyme, a hormone or a toxin.
  • the gene library comprises an enzyme activity of interest as described above.
  • the respective liquid enrichment media were prepared by mixing 100 ml of A+B+C+ 100 ml pectin or cellulose or starch and 600 ml sterile water.
  • the enrichments were used for preparation of a mixed enrichment library. 50 ml of each selected enrichment culture were centrifuged and the combined cell pellets used for gene library construction. The clones were obtained by screening of the library, distributed on amylase, arabinase, xylanase, galactanase and pectinase activity.
  • the cells from the cultures were washed with 0.9% NaCl and pooled into one tube .
  • DNA was extracted using method described by Pitcher et al . (Pitcher, D. G. , saunders,N.A. ,Owen;R.J. (1989) . Rapid extraction of bacterial genomic DNA with guanidium thiocyanate. Lett .Appl.Microbiol. , 8,151-156.). The DNA extraction recovered 170 ⁇ g high molecular weight DNA.
  • the DNA were trimmed to make blunt ends .
  • the reaction mixture was incubated at room temp, for 30 min, and 200 ⁇ l 1 x TE, pH 8.0, were added.
  • the mixture was extracted with 1 x phenol-chloroform, 1 x CIA, 0.1 vols 3 M NaOAc was added, pH 5.2, 2 vols 96 % EtOH were added, ppt on ice for 30 min, or overnight at -20°C and resupended in 16 ⁇ l H 2 0.
  • the end trimmed DNA was ligated into a fresh Eco RV digested pZero (Invitrogen) .
  • the ligation mixture was transformed into DH10B E.coli cells by electroporation and frozen in aliqouts corresponding to 300 zeocin resistant colonies.
  • the frozen alliqouts constitute the library no. 1.
  • the DNA was partially digested with the restriction enzyme Sau3A and the DNA was size-fractionated on a 1% agarose gel (Maniatis et al . ) .
  • the agarose containing the DNA corresponding to 3kb and upwards in size was cut from the agarose gel and the DNA was concentrated by further electrophoresis into a 1.2% agarose gel.
  • the DNA was isolated from the agarose piece using the GFX kit (Pharmacia) .
  • the Sau3A digested DNA was ligated into a fresh BamHI digested pZero-2 (Invitrogen) .
  • the ligation mixture was transformed into DH10B E. coli cells by electroporation and frozen in aliqouts corresponding to 300 kanamycin resistant colonies. The frozen aliqouts constitute the library no. 2.
  • the assay contains the following reagents:
  • 384 well In each well were 60 ⁇ l ( ⁇ 25ml/microplate) used as standard volume .
  • the cells were grown for 65 hours, and thereafter 50 ⁇ l of cells were pipetted into 150 ⁇ l assay substrate for 96 well plates or
  • Arabinase assay 20 contains the following reagents:
  • 384 well In each well were 60 ⁇ l used as standard volume.
  • the cells were grown for 65 hours, and thereafter 50 ⁇ l of cells were pipetted into 150 ⁇ l assay substrate for 96 well plates or
  • the assay contains the following reagents
  • the cells were grown for 65 hours, and thereafter 50 ⁇ l of cells were pipetted into 150 ⁇ l assay substrate for 96 well plates or 20 ⁇ l into 60 ⁇ l assay substrate for 384 well plates. The assay- plates were incubated over night at 50°C in a bag. Positive
  • Numbered black microtiter plates were filled with 150ml assay 20 mix* as described below using a Multidrop instrument. Subsequently, 50ml of cells were pipetted automatically into the assay plates using a Plate Mate pipetting station. Plates were left at room temperature (in the dark) for approximately 150-180 min., and subsequently read by a FPM-2 fluorescence 25 polarization reader using excitation-filter 485/22 and emission-filter 530/30. Positive clones were scored as a lowering of the polarization value which typically was from approximately 90mP to 50-70mP.
  • the assay contains the following reagents
  • 96 well In each well were 150 ⁇ l used as standard volume. 384 well: In each well were 60 ⁇ l used as standard volume. The cells were grown for 65 hours, and thereafter 50 ⁇ l of cells were pipetted into 150 ⁇ l assay substrate for 96 well plates or 20 ⁇ l into 60 ⁇ l assay substrate for 384 well plates. The assay- plates were incubated over night at 50°C in a bag. Positive reactions were observed as blue colour in the well.
  • Gene Library no . 2 was screened on LB agar plates containing 25 ⁇ g/ml kanamycin as the antibiotic selection marker and 0.03% AZCL-xyloglucan + 0.03% AZCL-galactan + 0.03% AZCL-amylose as enzyme substrates at 37°C. The formation of a blue halo around the colony indicates enzyme activity. The colony was restreaked onto LB plates containing each of the AZCl substrates to identify the enzyme activity. Three amylase positive clones were discovered.
  • Termite larvae (Neotermes castaneus) were acquired from BAM (Bundesweg fur Materialtechnisch für -Pr ⁇ fung,
  • Enrichment procedure The larvae were subsequently reared and fed on non-sterile plant materials, originating either from gymnosperms or angiosperms (monocot or dicot) , enabling enzymatic (endo- and exo-) digestion through plant cell wall degrading enzyme activity.
  • D ⁇ A preparation D ⁇ A preparations were made from such composite gut materials using commercially available D ⁇ A kits (FAST D ⁇ A- KitH, Bio 101 Inc, 1070 Joshua Way, California, US) . This high quality D ⁇ A material was used to prepare a genomic library, e.g. following a protocol as follows: digestion by Sau3A, fractionation and selection of specific size range, cloning in the Bacteriphage Lambda-Zap-Express (AH Diagnostic, originating from Stratagene, US) . Full protocols are given by the kit- manufacturers .
  • RNA preparation Total RNA preparations were made from said composite gut materials using commercially available RNA kits and public protocols, as e.g.
  • Identified hits more than 200 cellulase active clones were finally identified on HE Azur cross linked blue granule substrates from MegaZyme. PCR made directly from individual colonies (PCR procedures as indicated in relevant text books, using e.g. the polymerases available from Advanced Biotechnologies, Surrey, UK) was used to differentiate and group the hits. The primers used were based on recognition and amplification of the sense and antisense cDNA cloning plasmid pYes-2 (commercially available from Invitrogene, US) . At least four different sized functional genes were hereby identified.
  • Enrichment procedure The larvae were subsequently reared and fed on non-sterile protein-rich materials (as e.g. feather, hair and wool) . Further enrichment through dissection (optional) : The larvae were decapitated under a stereoscopic microscope after which the gut (including gut content) was selected and pooled together from several animals
  • DNA preparation DNA preps were made from such composite gut materials using commercially available DNA kits. This high quality DNA material was used to prepare a genomic library.
  • RNA preparation Total RNA preps were made from said composite gut material using commercially available RNA kits and protocols. The mRNA fraction was subsequently harvested. Based on this the corresponding cDNA prep was made. This was used to construct a cDNA library, representing the expressed proteins at the given time. Screening of the ⁇ enomic library enriched for DNA from those organisms specifically benefiting from the feeding conditions of the larvae under preparation: a plaque screening procedure was adapted to enzyme substrate holding plates, indicating which phages were holding an insert of a full functional gene encoding an enzyme with the protease activity of interest. Screening of the cDNA library enriched for high expression of proteins useful to degrade the feed given to the larvae : the protocol for expression cloning, as given in H.Dalb ⁇ ge, 1997 (FEMS Microbiology Reviews 21, 29-42) .
  • protease active clones could be identified by screening on substrate plates with AZCL-casein blue granules from MegaZyme. The protease hits could be further subdivided according to which types of protein bonds they specifically degrade.
  • Larvae of Melolontha vulgaris (Coleoptera) were collected from Danish habitats (Zealand) where the soil is rich in a very varied composition of plant debris. The larvae of this species are free living in soil, feeding for up to 3 years on plant materials
  • Enrichment procedure The larvae were subsequently reared and fed non-sterile non-specified plant debris of a very broad taxonomic composition. Further enrichment through dissection (optional) : The larvae were decapitated under a stereoscopic microscope after which the guts (including gut content) were selected and pooled together from several animals.
  • DNA preparation DNA preps were made from such composite gut material using commercially available DNA kits. This high quality DNA material was used to prepare a genomic library.
  • RNA preparation Total RNA preps were made from said composite gut material using commercially available RNA kits and protocols. The mRNA fraction was subsequently harvested. Based on this, the corresponding cDNA prep was made. This was used to construct a cDNA library, representing the expressed proteins at the given time.
  • a plaque screening procedure was adapted to enzyme substrate holding plates, indicating which phages were holding a (functional) gene insert encoding an enzyme with the plant cell wall degrading activity of interest .
  • Enrichment procedure The larvae were subsequently reared and fed non-sterile starch rich materials. Further enrichment through dissection (optional) the larvae were decapitated under a stereoscopic microscope after which the guts (including gut content) were selected and pooled together from several animals .
  • DNA preparation DNA preps were made from such composite gut material using commercially available DNA kits. This high quality DNA material was used to prepare a genomic library.
  • RNA preparation Total RNA preps were made from the composite gut material using commercially available RNA kits and protocols. The mRNA fraction was subsequently harvested. Based on this fraction the corresponding cDNA prep was made. This was used to construct a cDNA library, representing the expressed proteins at the given time.
  • a plaque screening procedure was adapted to enzyme substrate holding plates, indicating which phages were holding an insert of a functional gene, encoding an enzyme with a plant cell wall degrading activity of interest.
  • Enrichment procedure The cow was in the weeks prior to the sampling fed material of specific composition e.g. hey to enrich for cellulase and other plant cell wall degrading enzymes, cereal grains to enrich for amylase activity and soy to enrich for protease activities.
  • specific composition e.g. hey to enrich for cellulase and other plant cell wall degrading enzymes, cereal grains to enrich for amylase activity and soy to enrich for protease activities.
  • DNA preparation DNA preps were made from such composite rumen material using commercially available DNA kits. This high quality DNA material was used to prepare a genomic library.
  • RNA preparation Total RNA preps were made from the composite rumen material using commercially available RNA kits and protocols. The mRNA fraction was subsequently harvested. Based on this the corresponding cDNA prep was made. This was used to construct a cDNA library, representing the expressed proteins at the given time. Screening of the genomic library enriched for DNA from those organisms specifically benefitting from the feeding conditions of the larvae under preparation: a plaque screening procedure was adapted to be made on enzyme substrate holding plates, indicating which phages were holding an insert of a full functional gene, encoding an enzyme with the activity of interest .

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Abstract

L'invention concerne un procédé de création d'une banque de gènes à partir d'un groupe d'organismes prélevés dans l'environnement. Cette banque de gènes est enrichie en ADN codant pour un polypeptide présentant une activité d'intérêt. L'invention concerne également un procédé de sélection d'une séquence d'ADN d'intérêt à partir d'un groupe d'organismes prélevés dans l'environnement et une banque de gènes obtenue à partir d'un groupe d'organismes prélevés dans l'environnement enrichis avec un ADN codant pour un polypeptide doté d'une activité d'intérêt.
EP99948722A 1998-10-28 1999-10-14 Procede de creation d'une banque de genes Withdrawn EP1124948A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DK138898 1998-10-28
DKPA199801388 1998-10-28
PCT/DK1999/000553 WO2000024882A1 (fr) 1998-10-28 1999-10-14 Procede de creation d'une banque de genes

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EP1124948A1 true EP1124948A1 (fr) 2001-08-22

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EP (1) EP1124948A1 (fr)
JP (1) JP2002528075A (fr)
CN (1) CN1325445A (fr)
AU (1) AU6188699A (fr)
CA (1) CA2343878A1 (fr)
WO (1) WO2000024882A1 (fr)

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EP1756277B1 (fr) 2002-12-19 2009-12-02 Nuevolution A/S Procedes de synthese guidee a fonction et structure quasi-selectives
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CA2343878A1 (fr) 2000-05-04
AU6188699A (en) 2000-05-15
CN1325445A (zh) 2001-12-05
WO2000024882A1 (fr) 2000-05-04
JP2002528075A (ja) 2002-09-03

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