JP2003520578A - Methods for obtaining nucleic acids from environmental samples, resulting nucleic acids, and uses in the synthesis of novel compounds - Google Patents

Abstract

(57) Abstract The present invention relates to a method for producing a nucleic acid from an environmental sample, and more particularly to a method for obtaining a library of nucleic acids from a sample. The invention also relates to the nucleic acids of the nucleic acid libraries obtained by the method and their use in the synthesis of novel compounds, especially novel compounds of therapeutic interest. The present invention further relates to a novel means (eg, a novel vector) used in the method for obtaining the nucleic acid, and a novel method for producing the vector or the recombinant host cell containing the nucleic acid. Finally, the present invention relates to a method for detecting a nucleic acid of interest in a library of nucleic acids obtained by the method, the nucleic acid detected by the method, and the polypeptide encoded by the nucleic acid.

Description

Detailed Description of the Invention

The present invention relates to a method for producing nucleic acids from environmental samples, and more particularly to a method for obtaining a collection of nucleic acids from a sample. The invention also relates to the nucleic acids or populations of nucleic acids obtained by said method and their use in the synthesis of new compounds, in particular novel compounds of therapeutic interest.

[0002] The invention also relates to novel means (eg, novel vectors) for use in the above methods for obtaining nucleic acids, and novel methods for producing such vectors or recombinant host cells containing the nucleic acids of the invention.

The invention also relates to a method of detecting a nucleic acid of interest in a population of nucleic acids obtained by said method, and the nucleic acid detected by such a method and the polypeptide encoded by such a nucleic acid.

The invention also relates to nucleic acids obtained and detected by the above method, in particular nucleic acids encoding enzymes involved in the biosynthetic pathway of antibiotics such as β-lactams, aminoglycosides, heterocyclic nucleotides or polyketides, And an enzyme encoded by these nucleic acids, a polyketide produced by the means for expressing these nucleic acids, and finally a pharmaceutical composition comprising a pharmacologically active amount of the polyketide produced by the means for expressing such nucleic acids. Regarding things.

Since the discovery of streptomycin production by actinomycetes, the use of screening methods for metabolites produced by soil microorganisms has increased in the search for novel compounds of therapeutic interest, especially new antibiotics.

[0006] Such methods mainly separate the organisms of the soil microbiota and cultivate them in a specially adapted nutrient medium, which is then optionally subjected to one or more separation and / or purification steps beforehand. Detecting the pharmacological activity in the product present in the lysed cell lysate or in the culture supernatant.

Thus, methods for the in vitro isolation and culture of organisms that make up the telluric microflora have, to date, allowed the characterization of about 40,000 molecules. And about half of them show biological activity.

Major products such as antibiotics (penicillin, erythromycin, actinomycin, tetracycline, cephalosporins), anti-cancer agents, anti-cholesterolemia agents or pesticides have been characterized by such in vitro culture methods.

The majority of the therapeutically interesting products of microbial origin known to date (about 70%) are from actinomycetes, in particular of the genus Streptomyces. However, other therapeutic compounds, such as teicoplanin, gentamicin and spinosyn, have been found to be more difficult to culture in microorganisms of the genus, such as Micromonospora (
Micromonospora), Actinomadura (Actinomadur)
a), Actinoplanes, Nocardia
rdia), Streptosporongium (Streptosporongium)
), Kitasatosporia or Saccharomonospora.

However, it has been shown in practice that the characterization of new natural products synthesized by microorganisms of the soil microflora is still limited, in part due to the in vitro culture process. However, it usually causes selection of already known organisms.

Thus, methods for the in vitro isolation and culture of terrestrial microorganisms to identify novel compounds of interest have numerous limitations.

For example, in actinomycetes, the rate at which already known antibiotics are rediscovered is about 99%. In particular, fluorescence microscopy techniques allow the enumeration of more than 10 ¹⁰ bacterial cells per gram of soil, but 0.1% of these bacteria can be isolated after inoculation on medium. Only 1%.

With the help of DNA recombination kinetics technology, 12,000
It has been possible to show that 0 to 18,000 bacterial species can be contained in 1 g of soil, while non-eukaryotic microorganisms are currently Only 5000 species are listed.

Molecular ecology studies have made it possible to amplify and clone large numbers of 16S rDNA novel sequences from environmental DNA.

[0015]   The results of these studies have tripled the number of previously characterized bacterial phyla.

At present, bacteria are subdivided into 40 phyla, some of which consist exclusively of non-cultivable bacteria. These recent results demonstrate the widespread biodiversity of to date untapped microorganisms.

In recent research, there is a particular industrial interest (especially therapeutic interest).
Attempts have been made to overcome a number of obstacles to the utilization of soil microbiota biodiversity, including the step of in vitro culture prior to compound isolation and characterization.

For example, where appropriate, methods have been developed which include the step of extracting DNA from terrestrial organisms prior to the separation of the organisms contained in the soil sample.

After lysing the bacterial cells without prior culturing in vitro, the DNA thus extracted is cloned into a vector used for transfecting host organisms to obtain DNA from soil bacteria. Build the library.

These libraries of recombinant clones can be used to detect the presence of a gene encoding a compound of therapeutic interest or to detect the production of a compound of therapeutic interest by these recombinant clones. To do.

However, the methods described in the prior art for the direct utilization of soil microbiota DNA show disadvantages during the implementation of each of the above steps, which drawbacks are obtained and utilized. It is of a nature that considerably impairs the quantity and quality of possible genetic material.

The prior art relating to each of the steps for constructing a library of DNA from soil samples is described in detail below, together with the technical drawbacks identified by the applicant and overcome by the present invention.

[0023]   1. Process of extracting DNA from soil sample   1.1 Direct extraction of environmental DNA   This is essentially a reserve of the organisms in the sample relative to the environmental sample.
It is a method using a DNA extraction technique directly performed after the in situ cell lysis.

[0024] Such techniques have been used on samples derived from aqueous media from both fresh and sea water. They generally exist in free form or in the form of particles, consisting of large volumes of water filtration on various filtration devices, for example conventional membrane filtration, tangential or rotary filtration or ultrafiltration. A first step of pre-concentrating the cells to be treated.

The pore size is 0.22 to 0.45 mm, and pre-filtration is often required in order to avoid clogging due to treatment with a large volume.

In the second step, the harvested cells are lysed directly on the filter in a small volume of solution by enzymatic and / or chemical treatment.

This technique is described, for example, in Stein et al., 1996, Journal of Ba.
Cteriology, Vol. 178 (3): 591-599, which authors describe the marine plankton archaea (Archaebacte).
ria) and the cloning of the gene encoding the transcription elongation factor (EF2) and the ribosomal DNA.

Techniques for the direct extraction of DNA from soil or sediment samples have also been described and are based on physical, chemical or enzymatic cell lysis performed in situ.

For example, US Pat. No. 5,824,485 (Chromomame Corp)
describes the chemical lysis of bacteria directly to the sample by the addition of a guanidinium isothiocyanate-based hot cell lysis buffer.

International Patent Application WO 99/20799 (Wisconsin Alumni)
Research Foundation) describes a process of in situ cell lysis of bacteria using an extraction buffer containing protease and SDS.

Other techniques have been used, such as freeze-thawing a sample for several cycles and then high-pressure pressing the thawed sample. A technique of bacterial cell lysis using a series of steps of sonication, sonication and heat shock has also been used (P
icard et al. 1992).

However, the above-mentioned prior art techniques for direct extraction of DNA are very variable in their effectiveness in terms of quantity and quality.

For example, in situ chemical or enzymatic treatment of the sample is due to the selective resistance of various microorganisms inherent in the cell lysis process due to the heterogeneity of morphological features,
It has the disadvantage of lysing only certain categories of microorganisms.

For example, Gram-positive bacteria are resistant to treatment with heat SDS detergent, while virtually all Gram-negative cells are lysed.

Also, some of the above direct extraction protocols promote adsorption of extracted nucleic acids onto the mineral particles of the sample, significantly reducing the amount of available DNA.

Further, some of the prior art protocols disclose mechanical processing steps to lyse the microorganisms in the sample taken, such mechanical cell lysis steps being Systematically carried out in a liquid medium therein, it does not allow good homogenization of the starting sample in particulate form, which allows maximum availability for the diversity of the microorganisms present in the sample. Using glass beads
Milling tests have also been performed on crude soil samples, but the amount of DNA extracted was low.

The first step of in situ mechanical cell lysis in a liquid medium can be extracted DN
A negative effect on the amount of A has been observed in the present invention.

Also, the amount of DNA that can be directly used for cloning in the recombinant vector depends on the purification step after its extraction.

In the prior art, the extracted DNA is then, for example, by using polyvinylpolypyrrolidone or by precipitation in the presence of ammonium acetate or potassium acetate or by centrifugation on a cesium chloride gradient. , Or especially by a chromatographic technique on a hydroxyapatite support, an ion exchange column or a molecular sieve, or by an electrophoretic technique on an agarose gel.

The previously described DNA purification techniques, especially when combined with the above techniques for extracting environmental DNA, co-purify the inhibitory compounds from the initial sample, which are difficult to remove, with said DNA ( Co-purification).

Co-extraction of an inhibitory compound with the DNA inevitably leads to an increase in the number of purification steps, which leads to a considerable loss of initially extracted DNA and at the same time initially contained in the sample. Reduce the diversity and quality of the genetic material that was retained.

Another object of the invention is, on the one hand, to overcome the drawbacks of the pre-purification protocol and to develop a DNA purification process which allows to maintain an optimal level of diversity of DNA in the initial sample. , And on the other hand, was to quantitatively accelerate its production.

Most particularly, the qualitative and quantitative improvements to the purification of DNA are highest when it uses the combination of the direct DNA extraction method of the invention and the subsequent purification method, as explained below. Become.

[0044]   1.2. Indirect extraction of environmental DNA   Such a technique can be used to produce different species of terrestrial microbiota before the actual DNA extraction process.
It includes a first step of separating the product from the other constituents of the starting sample.

In the state of the art, the pre-separation of the microbial fraction from soil samples is usually by grinding it in a liquid medium (eg Waring Blender.
Or by using a device such as a mortar) to physically disperse the sample.

Chemical dispersion, for example dispersion on ion exchange resins or non-specific surfactants (
Dispersions using, for example, sodium deoxycholate or polyethylene glycol) have already been described. Whatever the dispersion method, solid samples should be suspended in water, phosphate buffer or saline solution.

After the physical or chemical dispersion step, it is possible to carry out centrifugation of the cells contained in the sample and on a density gradient allowing the separation of the sample particles, most soils It is understood that bacteria have a lower density than particles.

Alternatively, after the physical dispersion step, a low speed centrifugation step or a cell sluice step can be performed.

The DNA can then be extracted from the separated cells by any available cell lysis method and purified by a number of methods, including the purification methods described in Section 1.1 above. It is possible to In particular, to control the cell lysis,
It is possible to add the cells in low melting agarose.

However, the method described in the prior art known to the Applicant is such that undesired constituents of the starting sample which have a significant effect on the final quality and quantity of the DNA are found in the extracted DNA containing fraction. It is not satisfactory because it exists.

As will be described later, the present invention is presented to solve the technical problems found in the prior art methods.

[0052]   2. Molecular characterization of extracted DNA   I want to construct a DNA library from environmental samples (especially soil samples)
In some cases, the quality and diversity of the extracted and purified DNA source may be
It is convenient to check it before inserting it into the tractor.

The purpose of such molecular characterization of the extracted and purified DNA is to obtain a profile representing the proportion of different bacterial taxa present in the DNA extract. The molecular characterization of the extracted and purified DNA depends on whether the artifacts were introduced during the performance of the various extraction and purification steps, and, where appropriate, the original diversity of the extracted and purified DNA. , It is possible to determine whether or not to represent the microbial diversity that was originally present in the sample, especially the soil sample.

To the knowledge of the Applicant, the prior art utilizes quantitative hybridization methods using oligonucleotide probes specific for different bacterial groups applied directly to DNA extracted from the environment. To do.

Unfortunately, such an approach is relatively insensitive and does not allow detection of taxa or genera present in low abundance.

The prior art also describes quantitative PCR methods such as MPN-PCR or competitive quantitative PCR. However, these techniques have major drawbacks.

For example, MPN-PCR is cumbersome to perform due to the large number of dilutions and iterations, which makes it unsuitable for large numbers of samples or primer pairs.

Furthermore, competitive quantitative PCR is difficult to perform because it requires the construction of a competitor that is specific to the target DNA and that does not bias the competition itself or induce artifacts.

Therefore, the present invention allows for the rapid, convenient and reliable determination of the quality of pre-extracted and purified DNA and the determination of the value of constructing a library of clones prepared from this purified starting DNA. Derived from environmental samples
A method for prescreening the library of A is presented.

[0060]   3. For cloning DNA extracted and purified from environmental samples vector   In the prior art, cloning pre-extracted DNA from environmental samples
A number of vectors for doing so have already been described.

For example, according to the description of international patent application WO 99/20799, viral vectors, phages, plasmids, phagemids, cosmids, fosmids, BAC (bacterial artificial chromosome) type or bacteriophage P1 vectors, PAC type (
A bacteriophage P1-based artificial chromosome) vector, a YAC (yeast artificial chromosome) type vector, a yeast plasmid, or any other vector capable of stably maintaining and expressing genomic DNA can be used.

Example 1 of PCT patent application WO 99/20799 describes the construction of a genomic library by cloning into a BAC-type vector.

To the knowledge of the Applicant, DNA libraries derived from environmental samples are
Mating-type vectors have not yet been effectively produced, and such techniques were, for the first time, made available and reproducible to those skilled in the art by the teachings of the present invention.

[0064]   4. Host cell   In the prior art, it is derived from DNA extracted and purified from environmental samples.
Be used to house a vector containing a DNA insert
Thus, a large number of host cells have been described.

For example, PCT patent application WO 99/20799 describes a number of suitable host cells, for example Escherichia coli, in particular strain DH 10.
B or strain 294 (ATCC 31446, strain E. coli B, E. coli X 1776 (ATCC No. 31.537), E. coli (E.
E. coli DH5α and E. coli W3110 (ATCC No. 2)
7.325).

This PCT patent application also describes other suitable host cells, for example Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella (S).
almonella, Serratia, Schige
Lla), or a strain of Bacillus type, such as B. subtilis (B. subt)
ilis) and B. licheniformis
s), as well as Pseudomonas, Streptomyces or Actinomyces.
es) genus bacteria.

US Pat. No. 5,824,485 specifically discloses Streptomyces lividans (
Streptomyces lividans) strain TK66 or yeast cells such as Saccharomyces pombe.

[0068]   5. Characteristics of genes of interest in DNA libraries from environmental samples End   PCT patent application WO 99/20799 describes in each case a hemolysin-producing chromophore.
, Clones that hydrolyze esculin, or claws that produce orange pigment.
Belonging to the DNA library of B. cereus
Phenotypic identification of various clones that

Mutagenesis techniques based on the use of transposons encoding the phoA enzyme then made it possible to isolate mutagenized clones and to characterize the sequences leading to the observed phenotype.

Stein et al. (1996), supra, described specificity for ribosomal DNA to amplify DNA inserted into a vector carried by a clone of a marine plankton archaeal genomic DNA library. The use of primers and the identification of some coding sequences within the DNA so amplified is described.

Borschert S.D. Et al. (1992) refer to Bacillus subtilis (B
Bacillus subtilis using a pair of primers that hybridize to a conserved region of known peptide synthetases to identify one or more corresponding genes in the genome of A. subtilis.
s) genomic DNA library screening.

This technique made it possible to detect an approximately 26 kb chromosomal DNA fragment carrying the surfactin biosynthetic operon.

Kah-Tong S. Et al. (1997) describe the screening of a library of soil-derived DNA using primers that hybridize to conserved sequences of operons leading to the biosynthetic pathway of type II polyketides, PKS-
The identification of sequences belonging to the β gene in this DNA library is shown.
This reference also describes the construction of a hybrid expression cassette in which the sequence of the PKS-β subunit naturally found in the operon resulting in polyketide biosynthesis has been replaced with various similar sequences found in the DNA library. It has been described.

Similarly, the Hong-Fu et al. (1995) reference describes the construction of expression cassettes containing different open reading frames of the operon leading to polyketide biosynthesis, and such different expression cassettes. Is Streptomyces
It is artificially constructed by incorporating into the genome of Streptomyces coelicolor an open reading frame that is not found naturally together. This document shows that the combination of open reading frames from various bacterial strains in the artificial expression cassette is based on Bacillus subtilis and Bacillus cereus (Bac).
It has been shown to allow production of polyketides with relatively large antibiotic activity against Illus cereus) as well as various structural features.

Polyketides form part of a large family of natural products of varying structure with great diversity of biological activity. Examples of polyketides include tetrasacrine and erythromycin (antibiotic), FK506 (immunosuppressive agent), doxorubicin (anticancer agent), monensin (coccidial growth inhibitor) and avermectin (
Antiparasitic agents).

These molecules include acylthioesters (generally acetyl, propionyl,
It is synthesized by a multifunctional enzyme known as polyketide synthase that catalyzes repeated cycles of condensation between malonyl or methylmalonyl thioester). Each condensation cycle results in the formation of β-keto groups on the growing carbon chain, which may then optionally undergo one or more series of reduction steps.

Given the great clinical interest in polyketides, their common biosynthetic mechanisms, and the high degree of conservation observed among groups of genes encoding polyketide synthases, interest in the development of novel polyketides by genetic engineering. Is increasing.

For example, novel engineered polyketides such as mederhodin A or dihydrogranatirhodin have been genetically engineered. The majority of novel polyketide molecules obtained by genetic engineering are structurally very different from the corresponding natural polyketides.

Novel polyketides of interest, especially those having an increased level of antibiotic activity or a different spectrum of antibiotic activity compared to their natural homologs, which are broader than known polyketides or otherwise more selective, in particular, It is clear from the prior art that there is a need to obtain polyketides of therapeutic interest.

[0080]   As will be seen below, this need is partially met by the present invention.

DESCRIPTION OF THE INVENTION The present invention relates firstly to a method for constructing a library of DNA derived from environmental samples. Probably such samples include, by random listing, aqueous media (fresh water or seawater), soil samples (soil surface, subsoil or sediment), or eukaryotic samples containing associated microbiota, for example: , Samples derived from plants, insects or marine organisms and having an associated microflora.

The development of a method for constructing a library of DNA from environmental samples (especially soil samples) must be carried out in order to obtain a DNA library in which the content of the nucleic acid of interest meets the original set goals. Includes critical steps that must be optimized.

The first critical step is the nucleic acid initially contained in the sample (ie,
Primarily involves the extraction and subsequent purification of the nucleic acids contained in the various organisms of which the microbiota of this sample is composed.

[0084]   The quality of purification of the extracted DNA is a factor that determines the result obtained.

The second important step for constructing a library of nucleic acids from environmental samples is the assessment of the genetic diversity of the extracted and purified nucleic acids. Development of a process for convenient and reliable preliminary screening of extracted and purified DNA to ensure that the phylogenetic diversity of the organisms initially present in the starting sample is taken into account at least in part Determines the value of the use of extracted and purified initial DNA source for the construction of the nucleic acid library itself, or vice versa, the excess excess introduced at the time of extraction and purification of the nucleic acid. Effectively allows to decide not to continue building the nucleic acid library due to artifacts. It was also confirmed in the present invention that the quality of the insert introduced into the vector to construct the library is a determinant. For example, the use of restriction enzymes to cleave DNA extracted and purified from environmental samples may be of a nature that introduces artifacts or "bias" into the structure of the resulting insert. confirmed. In particular, DNA extracted from the soil or other environment that is mostly derived from non-culturable organisms is, of course, unknown in the content of G and C bases and, depending on the origin of these organisms. It is composed of various molecules.

The third critical step is the insertion of the extracted and purified nucleic acid into a vector capable of incorporating a nucleic acid of selected length, which on the one hand is given host cell Their transfection or integration into the genome, while allowing their expression in such host cells, as appropriate.

Where the goal achieved is in the cloning and identification of a complete operon that can lead to a complete biosynthetic pathway for compounds of industrial interest, especially those of pharmaceutical or agricultural interest, Vectors of interest will be those that can integrate large nucleic acids (ie, those of size greater than 100 kb).

Definitions For the purposes of the present invention, the terms “nucleic acid”, “polynucleotide” and “oligonucleotide” refer not only to DNA and RNA sequences, but more than two nucleotides in single- or double-stranded form It also means a hybrid RNA / DNA sequence.

The term “library” or “collection” is used herein to refer to a set of extracted and, if appropriate, purified nucleic acids derived from an environmental sample. A set of replacement vectors, each of the recombinant vectors of the set comprising a nucleic acid derived from said set of extracted and, if appropriate, purified nucleic acids, or said set of extracted and suitable In some cases, a set of recombinant host cells comprising one or more nucleic acids derived from a set of purified nucleic acids, said nucleic acid being carried on one or more recombinant vectors or within the genome of said recombinant host cells. Is incorporated into).

The expression “environmental sample” is, in random listing, a sample derived from an aqueous source, such as freshwater or seawater, or a ground sample derived from a surface layer of soil, a sediment or a lower layer of soil (subsoil), and 1 shows a sample of eukaryotes, which may be multicellular, from plants, marine organisms or insects that have an associated microflora, which constitutes the organism of interest.

In the present invention, the term “operon” means a set of open reading frames in which transcription and / or translation is co-regulated by a unique set of signals for regulating transcription and / or translation. To do. In the present invention, the operon may also contain said signals for regulating said transcription and / or translation.

For the purposes of the present invention, the expression “metabolic pathway” or “biosynthetic pathway” refers to a series of anabolic or catabolic biochemical reactions that result in the conversion of a first species into a second species. means.

For example, the antibiotic biosynthetic pathway is such that the major metabolite is an intermediate product of the antibiotic,
It then consists of a series of biochemical reactions that transform into antibiotics.

The term “regulatory sequence operably linked to a nucleotide sequence desired to be expressed” refers to the nucleotide sequence of interest desired to be expressed such that expression of the sequence of interest is possible. Means that a transcriptional regulatory sequence is located, and regulation of the expression depends on factors that interact with the regulatory nucleotide sequence.

According to another terminology, the nucleotide sequence of interest whose expression is desired is
It is also possible to be placed “under the control” of the transcription regulatory nucleotide sequence.

For the purposes of the present invention, the term “isolated” means that the biological material has been removed from its original environment, the environment in which it is naturally located. Indicates.

For example, a polynucleotide or polypeptide that naturally exists in an organism (virus, bacterium, fungus, yeast, plant or animal) has not been isolated. The same polypeptide, or polynucleotide, that is isolated from its natural environment, separated from the flanking nucleic acid with which it is naturally inserted into the organism's genome, has been isolated.

Such a polynucleotide may be contained within a vector and / or such a polynucleotide may be contained within a composition, and even in such cases It remains in isolated form. This is because the vector or composition does not constitute its natural environment.

The term “purified” does not require that the material be present in an absolutely pure form, unless other compounds are present. Rather, this is a relative definition.

The polypeptide or polynucleotide is in a purified form after the starting material has been purified by at least an order of magnitude, preferably by 2 or 3, preferentially by 4 or 5 orders of magnitude.

For the purposes of the present invention, the “% identity” between two sequences of nucleotides or amino acids is determined by comparing two optimally aligned sequences over a comparison window. You can

Therefore, portions of the nucleotide or polypeptide sequences within the comparison window may be added or deleted (eg, “gap”) relative to a reference sequence to obtain optimal alignment of the two sequences. ) (The reference sequence does not include these additions or deletions).

The percentage (%) determines the number of positions at which the same nucleobase or the same amino acid residue is observed with respect to those two comparative sequences, and then the identity between those two bases or amino acid residues. It is calculated by dividing the number of positions in which is present by the total number of positions in the comparison window and then multiplying the quotient by 100 to obtain the percent sequence identity.

The optimal alignment of those sequences for the comparison was Wisconsin G
genetics Software Package, Genetics C
computer Group (GCG), 575 Science Docto
r, Madison, Wisconsin package can be accomplished by computer using known algorithms.

For example, the percent sequence identity is determined using the default parameters B
LAST software (BLAST version 1.4.9, March 1996, 1
February 998 BLAST 2.0.4. And BLAST September 1998
2.0.6. ) Is used (SF Altschul et al.,
J. Mol. Biol. 1990 215: 403-410, S.M. F. Alts
chul et al., Nucleic Acids Res. 1997 25: 3389.
-3402) (see Altschul et al., "Request").
Blast search for sequences similar / homologous). The request sequence and the database used may be of peptide or nucleic acid nature and any combination is possible.

[0106]   Extraction and purification of nucleic acids from environmental samples   1. Direct extraction of nucleic acids   To obtain a library of organism-derived nucleic acids contained in soil samples
On the one hand, the various organisms in the sample become available for subsequent nucleic acid extraction steps.
It is important to create a condition that
Enables the highest mechanical cell lysis of organisms in the sample
Buffers used mainly for subsequent extraction steps (mainly genomic and plasmid DNA)
According to the present invention, it is important to have direct access to
Has been done.

It has been shown by the present invention that the highest availability of microbial-derived nucleic acids from soil samples, for example, was achieved by dry grinding of pre-dried soil samples to obtain microparticles. There is. Thus, Applicants have determined that drying the soil sample prior to any subsequent treatment results in a significant reduction in agglomeration of the coarse soil sample, resulting in fine particles when subjected to a suitable milling process. It was confirmed to promote its subsequent disintegration in morphology.

Surprisingly, Applicants have found that the fine particles of dried soil samples are preferred for the extraction of nucleic acids of optimal quality that may be characteristically representative of the genetic diversity of the organisms initially present in the starting soil sample. It has been shown that it also has the physical characteristics. In particular, the method for the direct extraction of nucleic acids of the present invention can be carried out using rare microorganisms (eg, certain rare Streptomyces
Ptomyces) or spore-forming microorganisms).

For the purposes of the present invention, the term “fine particles” of the soil sample is about 50 μm.
Particles from a sample having an average size of (i.e., 45-55 [mu] m on average).

In the present invention, the fine particles are obtained from a soil sample that has been pre-dried or pre-dehydrated and then ground to obtain fine particles having an average size of 2 μm to 50 μm, and the obtained fine particles are used as a liquid buffer medium. Resuspend.

Such a liquid buffer medium may consist of a nucleic acid extraction buffer, in particular a conventional DNA extraction buffer well known to those skilled in the art.

Milling of the soil sample into fine particles mechanically lyses most of the organisms present in the original soil sample, as well as organisms that are not cell lysed by this mechanical treatment are optimally subjected to subsequent chemical chemistry. And / or has the dual function of being available for the enzymatic cell lysis step.

Therefore, a first subject of the invention is a method for producing a population of nucleic acids from a soil sample containing organisms, wherein fine particles are obtained by grinding a pre-dried or pre-dehydrated soil sample, The production method comprises the first step (Ia) of suspending the fine particles in a liquid buffer medium.

In a fully preferred embodiment, the milling step is carried out using an apparatus with agate or tungsten beads or with an apparatus with a tungsten ring. These devices are preferred because the hardness of materials such as agate or tungsten significantly enhances the production of microparticles of the size specified above. For this reason, the use of grinding equipment with glass beads, which has been found to be much less efficient, would preferably not be selected or avoided.

Drying or classification of the soil sample can be done by any method known to those skilled in the art. For example, the crude soil sample can be dried at room temperature for 24-48 hours.

As already indicated, the liquid buffer medium may consist of a medium for extracting the DNA present in the microparticles. 50 mM Tris, 20 mM EDTA,
100 mM NaCl and 1% (weight / volume) polyvinylpyrrolidone at pH 9
． The extraction buffer known as TENP, each containing 0, will most preferably be used.

Further, in the method for producing a nucleic acid population from a soil sample, after the step of pulverizing a pre-dried or pre-dehydrated soil sample to obtain fine particles, a step I- of extracting nucleic acid present in the fine particles is performed. It is characterized by the continuation of (b).

It is the general view that extraction of the nucleic acid involves co-extraction of undesired soil constituents and / or compounds, thus requiring purification of the extracted nucleic acid, and Subsequent purification steps must be sufficiently selective to allow the removal of undesired soil constituents and / or compounds and result in low loss in the amount of pre-extracted DNA Need to give a sufficient yield.

The step of purifying the DNA extracted from the particulates of the soil sample which meet the above-mentioned selectivity and yield criteria comprises two successive chromatographic steps, a molecular sieve, each of which is combined with the extracted DNA. It has been shown according to the invention to include treating with the above chromatography and anion exchange chromatography.

According to another feature of the above method, after the step I- (b) of extracting the nucleic acid, the extracted nucleic acid is subjected to the following two chromatographic steps: -a solution containing the nucleic acid is molecularly Passing over a sieve and then recovering an elution fraction rich in nucleic acids, passing the elution fraction rich in nucleic acids on an anion exchange chromatography carrier,
Then, the purification step I- (c
) Continues.

The nature and sequence of the chromatographic steps are essential for good selectivity and good yield for the step of purifying pre-extracted DNA from pre-dried or pre-dehydrated soil sample microparticles.

In a very advantageous manner, the “molecular sieve” type chromatographic carrier in the nucleic acid purification step is Sephacryl® S400.
It consists of an HR type chromatographic carrier or a chromatographic carrier of equivalent properties.

In a fully preferred embodiment, the anion exchange chromatography carrier used in the second step to purify the extracted DNA is Elutip®.
Type of carrier or chromatographic carrier of equivalent properties.

A combination of step I- (a) for obtaining fine particles of the dried soil sample, I- (b) for extracting nucleic acid present in the fine particles, and I- (c) for purification by the above-mentioned chromatography step is combined. Thus, in the present invention, the DNA of the organism originally contained in the sample is directly extracted from the soil without prior purification, and at the same time, for example, humic acid found in prior art methods. It is possible to avoid co-extraction of soil contaminants such as.

Contaminants such as humic acid significantly interfere with the analysis and subsequent use of nucleic acids for which purification is desired.

[0126] Also, in the above method, the step I- (a) of obtaining microparticles of a soil sample to obtain a substantially complete population of genetic diversity of nucleic acids originally present in the soil sample.
It is possible to obtain a nucleic acid contained in an organism which is not mechanically lysed in. Thus, the particulates of the soil sample may be subjected to subsequent steps of a chemical, enzymatic or physical cell lysis treatment or a combination of chemical, enzymatic or physical treatments.

In a first aspect, the method of the present invention for producing a population of nucleic acids from a soil sample also comprises, after step I- (a): treating the soil suspension in a liquid buffer by sonication. , Can be characterized by a subsequent step of extracting and recovering the nucleic acid.

In a preferred embodiment, the treatment with ultrasound is carried out by the 600W Vibracell Ultrasonicator sold by Bioblock.
A titanium micro-point type device such as a device or a Cup Horn type sonicator is used.

In a fully preferred embodiment, the sonication step is carried out for 1 to 7 minutes.
Performed at a power of 5 W, which comprises successive cycles of sonication, which itself is carried out for 50% of the duration of each cycle.

In a second aspect, the method comprises, after step I- (a):-treating the soil suspension in a liquid buffer medium by sonication, -after sonication, lysozyme or acrosome. Mopeptidase (achromop
characterized by the steps of incubating the suspension at 37 ° C. in the presence of eptidase), adding SDS and then centrifuging and precipitating the nucleic acid, and recovering the precipitated nucleic acid. sell.

Preferably, the step of incubation with lysozyme and in the presence of achromopeptidase comprises a final concentration of each of those enzymes of 0.3 mg / ml,
It is preferably carried out at 37 ° C. for 30 minutes.

Preferably, the SDS is used at a final concentration of 1% at a temperature of 60 ° C. for an incubation time of 1 hour, followed by centrifugation and precipitation.

In a third aspect, the method for producing a population of nucleic acids from said soil sample also comprises, after step I- (a): a step of vigorous mixing (vortex) followed by a simple stirring step A step of homogenizing the soil suspension; a step of freezing and then thawing the homogeneous suspension; a step of sonicating the suspension after thawing; an ultrasonic treatment Afterwards, incubating the suspension at 37 ° C. in the presence of lysozyme and achromopeptidase, adding SDS, then centrifuging and precipitating the nucleic acid, and Characterized.

Preferably, the suspension of soil particulates is mixed on the vortex machine and then homogenized by gentle agitation on a rotary agitator for a duration of 2 hours,
It is then frozen at -20 ° C.

Preferably, after thawing and before the sonication step, the suspension is again stirred vigorously on a vortex machine for 10 minutes.

Needless to say, preferably, the nucleic acid extracted by the embodiment of the method for the direct extraction of nucleic acid is obtained after a first pass on the molecular sieve and subsequent chromatography on the molecular sieve. The elution fraction obtained is purified by a purification step consisting of a subsequent passage over an anion exchange chromatography carrier.

[0137]   2. Indirect extraction of nucleic acids   In the second embodiment of the method for producing a population of nucleic acids from an environmental sample according to the present invention,
The organisms contained in the sample from other large constituents of the sample
The environmental sample is subjected to a first treatment, which is of a nature that enables it.

This second embodiment of the method for producing a population of nucleic acids according to the invention facilitates the production of large nucleic acids which are virtually impossible to obtain with the first embodiment of the method according to the invention described above, and the microparticles The mechanical cell lysing step carried out to obtain also has the effect of physically degrading the nucleic acids in the soil sample or the nucleic acids contained in the organisms in the soil sample.

For the purpose of isolating and characterizing nucleic acids which at least partially comprise all of the coding sequences belonging to the same operon, which can direct the biosynthesis of compounds of industrial interest, the production of large nucleic acids has been carried out by the Applicant. Being researched.

Preferably, a second embodiment of the method of the present invention for producing a population of nucleic acids from a soil sample is performed to achieve nucleic acids of size greater than 100 kb, preferably greater than 200, 250 or 300 kb, and most preferably Is 400, 500
Or even obtain nucleic acids of size greater than 600 kb.

This second embodiment of the method for producing a population of nucleic acids from an environmental sample according to the invention consists of a combination of four consecutive steps intended to obtain a nucleic acid having the characteristics mentioned above.

If the environmental sample is a soil sample, the first step to obtain a suspension by dispersing the soil sample in a liquid medium is to cause a significant mechanical lysis of the cells. Nonetheless, it has been shown by the present invention to promote the availability of the organisms contained in the sample.

The first step of obtaining a dispersion of the soil sample makes the organisms in the sample accessible to the external medium and also allows partial dissociation of the organisms in the sample and the macro component. Therefore, it allows the subsequent separation of the organisms initially contained in the sample from the other constituents of this sample.

When the environmental sample is derived from, for example, a plant, marine organism or insect,
Pretreatment by milling is necessary to make the organisms of the concomitant microbiota available for subsequent steps of the process.

The method comprises the step of separating the organism from the other inorganic and / or organic constituents obtained above by centrifugation on a density gradient. The organisms thus separated are then subjected to a step of cell lysis and a subsequent step of extraction of the nucleic acid.

Surprisingly, the step of centrifugation on a density gradient makes it possible to separate the cells of the organism in the soil particles contained in the sample suspension. Indeed, it would have been expected that some of the cells would be carried away with the macroparticles in the gradient phase. Also, it has never been shown that centrifugation of soil samples over a density gradient allows populations of organisms representative of the biodiversity present in the starting samples to be found at the aqueous phase / gradient interface. Was not done. Because these organisms can vary greatly in volume, density and shape. It could theoretically be inferred that they are found in the aqueous phase, or in the aqueous phase / density gradient interface, or in the density gradient itself.

Therefore, organisms with densities below or above the density of the density gradient used (density of 1.2-1.5 g / ml, preferably 1.3 g / ml) cannot be recovered, As a result, one of skill in the art would expect that there would be a bias in the representative of effectively separated organisms, and thus in the diversity of the extracted nucleic acids.

Also, in one particular embodiment of the method, a step of germination of spores (especially of actinomycetes) is performed. The purpose is to significantly increase the amount of actinomycete DNA recovered.

The final step consists of purifying the nucleic acid thus extracted on a cesium chloride gradient.

Surprisingly, the purification of nucleic acids on the cesium chloride gradient allows a considerable or even complete removal of the substances making up the density gradient. This feature is a determinant for subsequent use of the purified nucleic acid. This is because it is known that the density gradient is a strong enzyme inhibitor that can appropriately inhibit the catalytic activity of the enzyme of the extracted nucleic acid into the vector.

In this second embodiment, the method of producing a population of nucleic acids from an environmental sample containing an organism of the present invention comprises: (i) obtaining a suspension by dispersing the environmental sample in a liquid medium, And then homogenizing the resulting suspension by gentle stirring, (ii) adding the organism to other inorganic and / or organic constituents of the homogeneous suspension obtained in step (i) From the components by centrifugation on a density gradient, (iii) cell lysing the microorganism separated in step (ii) and extracting the nucleic acid, (iv) purifying the nucleic acid on a cesium chloride gradient Including a series of steps.

Preferably, a suspension of the soil sample is obtained by dispersing the sample by milling with a device such as a Waring Blender or a device of equivalent characteristics. In a fully preferred embodiment, the Waring Blender
The sample suspension is obtained after 3 successive grinding operations, each lasting 1 minute in a device such as. Preferably, the ground sample is cooled in ice between each of the grinding operations.

Preferably, then Nycomed Pharma AS. Company (Oslo,
The organisms are separated from the soil particles by centrifugation on a "Nycodenz" type density cushion sold by Norway). The preferred centrifugation conditions are 4 ° C., 10,000 × g for 40 minutes, preferably it is
"Rotor TST 28.38" type vibration (s sold by tron
wing-out) in a rotor equipped with buckets.

The annulus of the organism located after centrifugation in the interphase between the upper aqueous phase and the lower Nycodenz phase is then removed, washed by centrifugation and the cell pellet is then taken up in a suitable buffer.

The step (iii) of cell lysis of the organism separated in the step (ii) can be performed by any method known to those skilled in the art.

Preferably, the cells are lysed in the presence of lysozyme and achromopeptidase in a 10 mM Tris-100 mM EDTA solution at pH 8.0, preferably at 37 ° C. for 1 hour.

The actual extraction of the DNA is preferably carried out by adding a solution of lauryl sarcosyl (1% of the final weight of the solution) in the presence of proteinase K and adding the final solution at 37 ° C. at 30 ° C.
It can be performed by incubating for a minute.

The nucleic acid extracted in step (iii) is then purified on a cesium chloride gradient.
Preferably, the step of purifying the nucleic acid on a cesium chloride gradient is performed, for example, by Kontr.
on 65.13 type rotor by centrifugation at 35,000 rpm for 36 hours.

In one particular aspect of the method of the present invention for producing a population of nucleic acids from a soil sample containing organisms, the nucleic acids consist predominantly, if not exclusively, of DNA molecules.

In another embodiment, the nucleic acid is added to an organism contained in an agarose block, or an organism separated on a density gradient is added to the agarose block to lyse cells (eg, chemical and / or enzymatic cell lysis). ) Can be collected after.

Another subject of the invention is the step II- (i of the method for producing a population of nucleic acids according to the invention.
It comprises a population of nucleic acids obtained in v) or consisting of the nucleic acids obtained in step (c) or the subsequent steps of the method for producing a population of nucleic acids of the invention.

[0162]   The invention also relates to a nucleic acid characterized in that it is comprised in said population of nucleic acids.

In a first aspect, such nucleic acids that make up the population of the invention are characterized in that they contain a nucleotide sequence encoding at least one operon or part of an operon.

Most preferably, such operons encode all or part of a metabolic pathway.

Example 9 is for Streptomyces streptomyces.
alboniger) strain and the cloning thereof into the shuttle cosmids pOS700I and pOS700R, respectively. In the DNA library prepared in the integrative vector pOS700I, it has been shown by the present invention that the new clone contains a nucleotide sequence belonging to an operon leading to the puromycin biosynthetic pathway. Similarly, 12 clones containing the nucleotide sequence of the operon leading to the puromycin biosynthetic pathway have been identified in a DNA library prepared in replicative vector pOS700R.

In particular, certain integrative and replicative cosmids of the resulting library may contain the entire 12 kb sequence of the operon that triggers the puromycin biosynthetic pathway after digestion with the restriction endonucleases ClaI and EcoRV. With fragments.

Thus, in another aspect, the nucleic acids of the invention at least partially comprise the nucleotide sequence of the operon that results in the puromycin biosynthetic pathway.

Example 2 below shows pBluescr starting from lindane contaminated soil.
The construction of a DNA library of the method of the invention in the ipt SK ^- vector is described.

The recombinant vector was transformed into Escherichia coli DH10.
B cells and then the transformed cells in the presence of lindane,
Cultured in a suitable medium. Screening of clones on transformed cells of the library made it possible to show that 35 out of 10,000 clones screened had the lindane degrading phenotype. Li within these clones
The presence of the nA gene was confirmed by PCT amplification with primers specific for this gene.

Therefore, in another aspect, the invention also relates to a nucleic acid containing a nucleotide sequence for a metabolic pathway that causes the biodegradation of lindane.

Therefore, as described above, the method for producing a population of nucleic acids from a soil sample containing the organism of the present invention, and the method for producing a population of recombinant vectors containing the constituent nucleic acids of the population of nucleic acids are within the operon. It has clearly been shown to be well suited for the isolation and characterization of the nucleotide sequences contained in.

Additional proof that the present invention may identify coding nucleotide sequences involved in biosynthetic pathways that are regulated in the form of operons is also described below.
This is the cloning and characterization of sequences encoding polyketide synthases involved in the polyketide biosynthetic pathway, which belong to a family of molecules typified by molecules of great therapeutic interest (particularly antibiotics). It is about.

The subject of the invention is therefore also the constituent nucleic acids of the population of nucleic acids according to the invention, characterized in that they comprise all of the nucleotide sequence coding for the polypeptide.

[0174]   In a first aspect, the constituent nucleic acids of the population of nucleic acids of the invention are of prokaryotic origin.

In a second aspect, the constituent nucleic acids of the population of nucleic acids of the invention are of bacterial or viral origin.

[0176]   In a third aspect, the constituent nucleic acids of the population of nucleic acids of the invention are of eukaryotic origin.

In particular, such nucleic acids are characterized in that they are derived from fungi, yeasts, plants or animals.

[0178]   Molecular characterization of a population of nucleic acids extracted from soil   Extraction and purification from environmental samples as described in the prior art section
To overcome various technical drawbacks for characterizing a library of isolated DNA
To this end, the Applicant has characterized the nucleic acids obtained from said method qualitatively and semi-quantitatively.
We have developed a simple and reliable method for doing this.

Accordingly, the method of the present invention universally amplifies a 700 bp fragment located within the sequence of 16S ribosomal DNA, and then hybridizes the amplified DNA with oligonucleotide probes of various specificity, Finally, it consists in comparing the hybridization intensity of the sample to an external calibration range of DNA of known sequence or origin.

Amplification prior to hybridization with the oligonucleotide probe allows quantification of relatively rare microbial genera or species. Furthermore, amplification with universal primers allows the use of a wide array of oligonucleotide probes during the hybridization.

The subject of the present invention is therefore also a method for measuring the diversity of nucleic acids contained in a population of nucleic acids, in particular of a nucleic acid derived from an environmental sample, preferably a soil sample, which comprises: Contacting the nucleic acids of the population of nucleic acids with a pair of oligonucleotide primers that hybridize to any sequence of bacterial 16S ribosomal DNA, performing at least three amplification cycles, one oligonucleotide probe or a plurality of oligonucleotide probes Oligonucleotide probes (each probe is a 16S ribosomal DN common to the kingdom Bacteria, order, subclass or genus)
Hybridizing specifically to the sequence A)), and detecting the amplified nucleic acid, if appropriate, the results from the detection step are used for the nucleic acid of known sequence constituting the calibration range. A method comprising a step of comparing with a detection result when a probe or a plurality of the probes described above is used.

Preferably, the first pair of primers that universally hybridize to the conserved region of the gene for 16S ribosomal RNA is each primer FGPS 612 (
SEQ ID NO: 12) and FGPS 669 (SEQ ID NO: 13).

A second embodiment of the preferred pair of primers of the present invention is the universal primer 6
It consists of a pair of 3f (SEQ ID NO: 22) and 1387r (SEQ ID NO: 23).

In one particular embodiment of the method for measuring nucleic acid diversity of a nucleic acid population, prior to the step of hybridizing with an oligonucleotide probe specific for a particular bacterial kingdom, order, subclass or genus, An amplification step using universal primer pairs can be performed on a population of recombinant vectors in which nucleic acids from the population of nucleic acids under consideration have been inserted.

Such methods of measuring the diversity of nucleic acids contained within a population are particularly applicable to populations of nucleic acids obtained in accordance with the teachings herein.

Therefore, Example 3 is a method for producing a population of nucleic acids from a soil sample containing organisms, wherein the soil sample is dispersed prior to separation of the cells on a Nycodenz gradient and the cells are The method is detailed, which involves the steps of lysing and then indirect extraction of the DNA by purifying the DNA on a cesium chloride gradient.

The population of nucleic acids thus obtained is used as it is, or in the form of an insert in a cosmid-type vector, in an amplification method using said universal primers for 16S rDNA and then said The amplified DNA was subjected to a step of detection using oligonucleotide probes of sequences SEQ ID NO: 14 to SEQ ID NO: 21 shown in Table 4.

The results show that a method of producing a population of nucleic acids starting from a soil sample containing an organism of the present invention comprises a method for producing more than 14% of the total terrestrial microflora of DNA (ie 2 × 10 6 / g soil) ⁸ cells), while the total microbial flora that can be cultivated represents only 2% of the total microbial population.

To measure the phylogenetic diversity of the population of nucleic acids produced according to the invention, 47 sequences of the 16S rRNA gene were isolated and sequenced. These sequences correspond to the nucleotide sequences of SEQ ID NO: 60 to SEQ ID NO: 106, respectively.

Nucleic acids comprising the sequences SEQ ID NO: 60 to SEQ ID NO: 106 also form part of the invention and are at least 99%, preferably 99.5% of the nucleic acids comprising the sequences SEQ ID NO: 60 to SEQ ID NO: 106. The same applies to nucleic acids having a nucleic acid identity of% or 99.8%.
Such sequences can be used, inter alia, as probes to screen clones of a DNA library and thereby bring the clones of the library into close proximity to the coding sequence of interest. It can be used as a probe to identify clones containing potential sequences (eg, sequences encoding antibiotic metabolites such as enzymes involved in the biosynthetic pathway of polyketides).

The sequence of 16S rRNA from the DNA library obtained according to the invention,
RDP database (Maidak BL, Cole JR, Park)
er C. T. , Garrity G .; M. , Larsen N. et al. , Li B.
, Liburn T .; G. McCaughey, M .; J. , Olsen G.
J. , Overbeek R .; , Pramanik S .; , Schmidt T
． M. , Tiedje J .; M. , Woese C .; R. (1999) "A n
ew project of the RDP (Ribosomal Data)
base Project) "Nucleic Acid Research
Vol. 27: 171-173), the nucleic acid contained in the population of nucleic acids of the present invention is α-proteobacteria, β-proteobacteria, δ-proteobacteria, γ-proteobacteria, actinomycetes, And that it was derived from a genus associated with acidobacterium. These results, shown in the phylogenetic tree of Table 7 and FIG. 7, explain the large phylogenetic diversity of the nucleic acids contained within the DNA libraries obtained by the method of the invention.

[0192]   Cloning and / or expression vector   Each of the nucleic acids contained in the population of nucleic acids obtained according to the invention is cloned
And / or can be inserted into an expression vector.

For this purpose, any type of vector known in the prior art, eg viral vector, phage, plasmid, phagemid, cosmid, fosmid, BAC type vector, P1 bacteriophage, BAC type vector, YAC. Any type of vector, yeast plasmid or any other vector known to the person skilled in the art can be used.

Vectors which allow stable expression of the nucleic acids of the DNA library will be advantageously used in the present invention. For this purpose, such a vector preferably comprises transcriptional regulatory sequences operably linked to the genomic insert and allowing initiation and / or regulation of the expression of at least part of the DNA insert.

As can be seen from the above, the present invention provides the step II- (iv) or the step I- (c
) Or a nucleic acid obtained in any other subsequent step of the method for producing a population of nucleic acids from a soil sample containing an organism of the invention, characterized in that it is inserted into a cloning and / or expression vector. It relates to a method for producing a population of replacement vectors.

Prior to their insertion into cloning and / or expression vectors, the constituent nucleic acids of the population of nucleic acids according to the invention are subjected to electrophoresis on an agarose gel, for example by electrophoresis after appropriate digestion with restriction endonucleases. Can be separated according to the size of.

In another embodiment, the average size of the constituent nucleic acids of a population of nucleic acids of the invention is determined by performing a step of physical disruption prior to their cloning and / or their insertion into an expression vector. It is possible to have a uniform size.

Such a step of physical or mechanical disruption of nucleic acids may be performed in solution at a diameter of about 0.4 m.
It may consist of continuously passing these nucleic acids through m metal channels (eg channels of a syringe needle having such a diameter).

[0199]   The average size of the nucleic acid may in this case be 30-40 kb long.

Construction of a preferred vector for the present invention is shown schematically in Figure 25 (conjugated integrated cosmid) and Figure 26 (integrated BAC).

Cloning and / or expression vectors that can be advantageously used to insert the nucleic acids contained within the DNA libraries or populations of the present invention are, in particular, EP 0 350 341 and US Pat. No. 5 688 689. The vector described in 1. above, and such a vector is particularly suitable for transformation of an actinomycete strain. In addition to the insert DNA sequence, such a vector contains the attachment sequence att and a DNA sequence ( int sequence) encoding an integrase functional in the actinomycete strain.

However, it is acknowledged by the present invention that certain cloning and / or expression vectors have drawbacks and their theoretical functional capacity was not achieved in practice.

Therefore, in prior art vectors, in particular in European patent EP 0 350 34
It was found that the integration system contained in the vector described in 1 does not actually allow good integration of the DNA insert from the library into the bacterial chromosome.

Starting from the hypothesis that the functional defect of the integration of such vectors into the bacterial chromosome was due to the defective expression of the integrase gene present in these vectors. First attempted to increase expression of the integrase gene by replacing the initiating transcription promoter with a transcription promoter that could significantly increase the number of integrase transcripts.

The results were disappointing and the function of integration of these vectors into the chromosome was not improved.

Surprisingly, according to the invention, the problem with integrase expression contained in this family of integrative vectors lies not in the amount of transcript expression, but in the stability of the transcript. Was shown.

According to the second hypothesis, the applicant was able to show that the lack of stability of the integrase transcript is caused by the lack of termination of transcription of the corresponding messenger RNA.

Therefore, Applicants have inserted a termination site located downstream of the integrase coding sequence of the vector in order to obtain a messenger RNA of a given size. The insertion of an additional termination signal downstream of the nucleotide sequence encoding the integrase of the vector made it possible to obtain a family of cosmid-type and BAC-type integrative vectors.

[0209] Preferably, the termination site is located downstream of the attachment site att .

The Applicant has also found that a novel conjugative vector and a novel replicative vector of the cosmid type and a novel BAC type that can be advantageously used for inserting the constituent nucleic acids of the population of nucleic acids obtained by the method of the invention. A mating type vector was developed.

If an average size DNA fragment insertion is desired, a cosmid-type vector capable of accommodating an insert having a maximum size of about 50 kb is preferably used.

Such a cosmid vector inserts in particular the constituent nucleic acids of the population of nucleic acids obtained by the method according to the invention comprising the first step of direct DNA extraction by mechanical cell lysis of the organisms contained in the initial soil sample. Suitable to do.

Large nucleic acids, in particular larger than 100 kb or even 200,300
, If insertion of a nucleic acid larger than 400, 500 or 600 kb is desired,
BAC type vectors which can accommodate DNA inserts of such size are preferentially used.

Such vectors of BAC type, in particular, the first step consists of the pre-separation of the organisms contained in the initial soil sample and the indirect extraction of the DNA by removal of the large constituents from the soil sample. It is suitable for inserting the constituent nucleic acids of the nucleic acid population obtained by the method of the invention.

In particular, BAC-type vectors are advantageously used for inserting large nucleic acids which at least partly contain the nucleotide sequence of the operon.

Therefore, the method for producing a population of recombinant cloning and / or expression vectors according to the invention is also characterized in that the cloning and / or expression vector is of the plasmid type.

In another aspect, such a method is characterized in that the cloning and / or expression vector is of the cosmid type.

In the first aspect, it may be a cosmid that is replicative in E. coli and integrative in Streptomyces. A fully preferred cosmid corresponding to such a definition is Example 3.
POS7001 described in 1.

In yet another aspect, the cosmid vector is Streptomyces (St
reptomyces) are mating and embedded.

[0220] Generally, cosmid-type or BAC-type conjugative vectors containing a unit recognized by a cellular enzyme device known as "conjugation origin" in the nucleotide sequence are laborious transformation techniques that are difficult to automate. Used in any case where you want to avoid relying on.

For example, when a vector originally retained by E. coli cells is to be transfected into Streptomyces cells, Streptomyces protoplasts are usually transformed. Before the process, Escherichia coli (Escherichia coli)
) A step of recovering the recombinant vector contained in the cell and purifying it is required.
In order for each E. coli clone to have the opportunity for its expression, 1000 E. coli (Esc) into Streptomyces.
about 8 for transfection of an assembly of H.
It is generally believed that production of 000 clones is required.

Conversely, the step of transfection by conjugating the vector carried by E. coli into Streptomyces cells requires an equal number of clones of each of those microorganisms, The conjugation step occurs "clone to clone" and further does not involve the technical problems associated with introducing genetic material by transformation of protoplasts, for example in the presence of polyethylene glycol.

In order to optimize the construction of the DNA library in Streptomyces, a novel cosmid-type and BAC-type conjugation vector of the nature that allows maximum efficiency of the conjugation process was developed in the present invention. did.

In particular, the novel mating type vector of the present invention is constructed by placing a selectable marker gene at the end of the DNA of the vector introduced into the recipient bacterium. This improvement over prior art conjugative vectors allows positive selection of only those bacteria which received all of the vector DNA and therefore all of the insert DNA of interest.

A preferred cosmid of the present invention that is conjugative and integrative in Streptomyces is the cosmid pOSV3 described in Example 5.
03, pOSV306 and pOSV307.

In another aspect, the method of producing a population of recombinant vectors of the present invention comprises:
E. E. coli) and Streptomyces. Such a cosmid is preferably the cosmid pOS700R described in Example 6.

In yet another aspect, the method can be performed with cosmids that are replicative in E. coli and Streptomyces and conjugative in Streptomyces.

Such replicative and conjugative cosmids can be obtained from the replicative cosmids of the invention by inserting a suitable source such as RK2 described in Example 5 for the construction of vector pOSV303. You can

In another advantageous embodiment of the method for producing a population of recombinant vectors according to the invention:
BAC type cloning and / or expression vectors are used.

In the first aspect, the BAC-type vector is Streptomyces (Strep).
embedded type and mating type in

In a fully preferred embodiment, Streptomyces.
es) such a BAC vector which is integrative and conjugative is the vector BAC pOSV403 described in Example 8, otherwise the BAC pMBD-1, pMBD-2, pMBD-3 described in Example 15. pMBD-4, pM
BD-5 and pMBD-6.

The subject of the invention is also the following recombinant vectors: a) a vector containing the constituent nucleic acids of the nucleic acid population of the invention, b) a restriction endonuclease for the DNA fragment to be inserted, as already described. It is a recombinant vector characterized by being selected from vectors obtained by a method of avoiding involvement of action.

[0233]   In a fully preferred embodiment, the invention also provides the following vector: ・ Cosmid pOS700I, ・ Cosmid pOSV303, ・ Cosmid pOSV306, ・ Cosmid pOSV307, ・ Cosmid pOS700R, -Vector BAC pOSV403, -Vector BAC pMBD-1, -Vector BAC pMBD-2, -Vector BAC pMBD-3, -Vector BAC pMBD-4, -Vector BAC pMBD-5, -Vector BAC relates to a vector selected from pMBD-6.

The invention also relates to a population of recombinant vectors obtained by any one of the methods of the invention.

[0235]   Method for producing recombinant cloning and / or expression vector of the present invention   Intravector to produce recombinant cloning and / or expression vectors
The usual techniques for inserting DNA into the
Incubate the restriction endonuclease with both
By making compatible ends between the DNA to be inserted and the vector DNA,
Of the two DNAs before the final ligation step, which allows the production of the recombinant vector.
It includes a first step to allow assembly.

However, such conventional techniques have notable drawbacks, especially when it is desired to insert large nucleic acids into cloning and / or expression vectors.

In particular, the pre-action of restriction enzymes on the DNA fragments intended to be inserted into the vector tends to significantly reduce the size of this DNA prior to its insertion into the vector. Needless to say, the DN before its insertion into the vector
A significant reduction in the size of A is due to all of the coding sequence and, in some cases,
If one wishes to clone a large piece of DNA which tends to also contain regulatory sequences for the operons that are expressed to constitute the complete biosynthetic pathway of metabolites of industrial interest (particularly compounds of therapeutic interest) This is a particularly disadvantageous situation.

In order to overcome the drawbacks of the prior art, 2 for producing a recombinant cloning and / or expression vector in which no restriction endonuclease is used on the inserted DNA prior to its introduction into the vector. Two methods have been developed in the present invention. Accordingly, such methods clone long DNA fragments that tend to at least partially contain all of the coding sequences, and, where appropriate, also the regulatory sequences of the complete operon responsible for the biosynthetic pathway. Good enough for.

In a first aspect, one method for producing the cloning and / or expression vector of the invention is that the insertion of the nucleic acid into the cloning and / or expression vector comprises: Using a suitable restriction endonuclease And cleaving the cloning and / or expression vector at a selected cloning site, adding a first homopolymeric nucleic acid at the free 3'end of the opened vector, inserted into the vector Adding a second homopolymer nucleic acid having a sequence complementary to the first homopolymer nucleic acid at the free 3'end of the nucleic acid; hybridizing the first and second homopolymer nucleic acids having complementary sequences to each other Thereby assembling the nucleic acid of the vector and the nucleic acid: -closing the vector by ligation And wherein the Mukoto.

[0240]   Such a method is described in Examples 10 and 13 below.

[0241]   Preferably, the method may include the following features separately or in combination: The first homopolymeric nucleic acid is of poly (A) or poly (T) sequence; The second homopolymer nucleic acid is of poly (T) or poly (A) sequence.

In a fully preferred embodiment, the homopolymeric nucleic acid has a length of 25-100 nucleotide bases, preferably 25-70 nucleotide bases.

The above-mentioned method for producing a recombinant cloning and / or expression vector comprises BAC.
It is particularly suitable for the construction of DNA libraries in vector types. Therefore, in one advantageous embodiment of the method for producing a recombinant vector as described above, the method is also such that the size of the inserted nucleic acid is at least 100 kb, preferably at least 200, 300, 400, 500 or 600 kb. It is characterized by

Therefore, such a production method is particularly suitable for inserting a nucleic acid contained in the nucleic acid population obtained by the method of the present invention.

In order to allow the insertion of large DNA fragments into cloning and / or expression vectors, it is possible to dispense with the use of restriction endonucleases for the DNA intended for insertion into the vector. Method was developed in the present invention.

Such a method for producing a recombinant cloning and / or expression vector according to the invention comprises the steps of: inserting a nucleic acid into the cloning and / or expression vector: removing overhanging 3 ′ sequences and overhanging 5 ′ sequences. To create blunt ends on the ends of the nucleic acids of the population by filling in. Cleaving the cloning and / or expression vector at selected cloning sites using appropriate restriction endonucleases. A step of adding a complementary oligonucleotide adaptor, a blunt end is created at the end of the vector nucleic acid by removing the overhanging 3 ′ sequence and filling in the overhanging 5 ′ sequence, and then reconstituting the vector Dephosphorylating the 5'end to prevent cyclization, ligating the population of nucleic acids into the vector Characterized in that it comprises a step of further insertion.

Preferably, removal of the overhanging 3'sequence is performed using an exonuclease such as Klenow enzyme.

Preferably, the fill-in of the overhanging 5'sequence is performed using a polymerase, most preferably T4 polymerase, in the presence of four nucleotide triphosphates.

As described above, the method for producing a recombinant cloning and / or expression vector by removing the protruding 3 ′ sequence and filling in the protruding 5 ′ sequence is carried out by constructing a DNA library from a cosmid type vector. Especially suitable for.

[0250]   Such a method for obtaining recombinant vectors is described in Example 12.

In one particular method for producing the recombinant vector of the present invention, an oligonucleotide containing one or more rare restriction sites is introduced at the cloning site of the DNA to be inserted according to the teaching of Example 10. Add to vector. This addition of oligonucleotide facilitates the subsequent recovery of the insert, without its cleavage.

[0252]   Host cell   Transfection or transformation with a nucleic acid or recombinant vector of the invention
Any type of host cell can be used for
Well-characterized nuclear host cell, physiological, biochemical and genetic characteristics
A host cell whose culture conditions for the production of its metabolites are well known and
Host cells that can be easily cultured on a scale are preferably used.

Preferably, the host cell that receives the nucleic acid or recombinant vector of the invention is phylogenetically close to the donor organism originally contained in the environmental sample from which the nucleic acid was derived.

In the most preferred embodiment, the host cells of the invention should have similar or at least close codon usage in the donor organism originally present in the environmental sample, especially the soil sample.

The size of DNA fragments that tend to retain the desired nucleotide sequence of interest can vary. Thus, a gene-encoded enzyme with an average size of 1 kb can be expressed using a small size insert, while the expression of secondary metabolites is much greater than that of a much larger fragment (eg, , 40kb-1
00 kb, 200 kb, 300 kb, 400 kb or more than 600 kb) will be required to be maintained in the host organism.

Therefore, Escherichia coli host cells represent a preferred option for the cloning of large DNA fragments.

In a most preferred embodiment, the Escherichia coli strain known as DH10B and described in Shizuya et al. (1992), with optimized protocols for cloning into a BAC vector, is used. Will

However, to construct the DNA library of the present invention, E. coli (Esch
other strains of E. coli, such as E. coli Sur
e, E. coli DH5α or E. coli 294 (A
The TCC No. 31446) strain may be used to advantage.

Furthermore, it is possible to construct a DNA library by transfecting E. coli cells with the recombinant vector of the present invention.
Cillus), Thermotoga, Corynebacterium (
Corynebacterium, Lactobacillus
US) or PCT patent application WO 99/20799 describes the expression of various prokaryotic genes such as Clostridium.

In general, E. coli host cells, in all cases, constitute a transient host in which the recombinant vectors of the present invention may be effectively maintained, and in which the genetic material is easily manipulated and stable. Can be housed in.

In addition, for the purpose of expressing the widest possible molecular diversity, Bacillus (Baci)
llus, Pseudomonas, Streptomyces, Myxococcus, Aspergillus nidulans, or a host cell of Neurospora, such as Neurospora cells of Neurospora crassa. Can be done.

In addition, Streptomyces lividans (Streptomyces liv)
idans cells have been used successfully, and E. coli (Escherichia)
It has been shown by the present invention that an expression system which is complementary to E. coli can be constructed.

Streptomyces lividans
ns) constitutes a model for studying Streptomyces genetics and has been used as a host for the heterologous expression of numerous secondary metabolites. Streptomyces
lividans) is Streptomyces kericolor (Streptom)
yces coelicolor), Streptomyces griseus (Str)
eptomyces griseus), Streptomyces frazier (St
reptomyces fradiae) and Streptomyces griseochromogene (Streptomyces griseochromogene)
complex biosynthetic pathways (eg, polyketide biosynthetic pathways or pathways for the biosynthesis of non-ribosomal polypeptides that represent a highly diverse class of molecules), as well as other actinomycetes such as s). Have regulatory systems and precursor molecules required for expression of all or part of

Streptomyces lividans
ns) also has the advantage of accepting foreign DNA with high transformation efficiency.

Therefore, the present invention also relates to a recombinant host cell containing the nucleic acid of the present invention, which is a constituent of the nucleic acid population produced by the method of the present invention, or a recombinant host cell containing the above recombinant vector.

In the first aspect, it may be a recombinant host cell derived from a prokaryote or a eukaryote.

Preferably, the recombinant host cell of the present invention is a bacterium, most preferably a bacterium selected from E. coli and Streptomyces.

In another aspect, the recombinant host cell of the invention is characterized in that it is a yeast or a filamentous fungus.

The present invention is also a population of recombinant host cells, each host cell of the population being produced by a method of producing a population of nucleic acids from a soil sample containing an organism as described above. It relates to a population characterized in that it comprises nucleic acids derived from the population of nucleic acids.

The invention also relates to a population of recombinant host cells, each of the constituent host cells of said population comprising a recombinant vector of the invention.

Due to the large size of the insert, it is necessary to obtain maximum transformation efficiency. To this end, pSAM2 is used to facilitate site-specific integration of the vector.
Streptomyces lividans that expresses integrase constitutively
Recipient strains of Ptomyces lividans) are preferred. In this case, the int gene under the control of a strong promoter is integrated in the chromosome. Overproduction of integrase does not induce any excision phenomenon (Raynal et al., 1998).

If the insert does not contain a gene for antibiotic resistance produced,
Alternatively, if this gene is not expressed or is expressed only to a low extent, the production of new metabolites from the insert is not compatible with Streptomyces (Strep).
could be toxic to toxicants). The ability of various genes to allow it to resist antibiotics produced by Streptomyces ambofaciens has been studied (Gourmelen et al., 1998; Pernodet et al., 1999).
). Some of these genes encode ABC type transporters that tend to confer broad spectrum resistance. These genes are Streptomyces
It can be introduced into a Streptomyces lividans host strain and overexpressed in the host strain.

Conversely, antibiotic-sensitive strains can be used to detect the presence of resistance genes within the library (Pernodet et al., 1996). Particularly in antibiotic-producing microorganisms, these resistance genes are often associated with genes in the antibiotic biosynthetic pathway. Selection of resistant clones may facilitate the first selection prior to more complex testing for the detection of novel metabolites produced by the clone.

[0274]   Isolation and characterization of a novel nucleotide sequence encoding a polyketide synthase. Ke   In the present invention, nucleic acid inserts derived from the nucleic acid population produced by the method of the present invention are
Host cells with a population of recombinant vectors, each containing
After that, a population of recombinant host cells was obtained.

More specifically, the DNA fragment obtained by the method of the present invention (in which case the step of indirect extraction of DNA from the organism contained in the soil sample is carried out) is first cloned into the integrated cosmid pOS700I. did.

The step of inserting the DNA fragment into the integrated cosmid pOS700I was performed according to the method of the present invention. In this case, the homopolymer polynucleotide tail poly (A
) And poly (T) were added to the 3 ′ end of the vector nucleic acid and the inserted DNA fragment, respectively.

The recombinant vector constructed in this way was encapsulated in a lambda phage head,
The resulting phage was used to transform E. coli (E. c.
oli) infected cells.

A library of approximately 5000 Escherichia coli clones was obtained.

This library of clones was screened with a pair of primers specific for the nucleotide sequence encoding the type I PKS enzyme, also known as β-ketoacyl synthase, an enzyme involved in the polyketide biosynthetic pathway.

Here, polyketides constitute a very structurally diverse chemical category containing a large number of pharmaceutically interesting molecules such as tylosin, monensin, vermectin, erythromycin, doxorubicin or FK506. Is recollected.

Polyketides are synthesized by the condensation of acetate molecules by the action of an enzyme known as polyketide synthase (PKS). There are two types of polyketide synthases. Type II polyketide synthases are commonly involved in the synthesis of polycyclic aromatic antibiotics and catalyze the repeated condensation of acetate units.

Type I polyketide synthase is involved in the synthesis of macrocyclic or macrolide polyketides and constitutes a modular multifunctional enzyme.

Given their therapeutic interest, there is a need in the state of the art to isolate and characterize novel polyketide synthases that can be used in the production of new pharmaceutical compounds, especially novel pharmaceutical compounds with antibiotic activity. ing.

Screening of the library of recombinant clones described above using PCR primers that selectively amplify the nucleotide sequence encoding type I polyketide synthase was carried out using DNA containing the nucleotide sequence encoding the novel polyketide synthase.
It was possible to identify recombinant clones containing the insert. The nucleotide sequences encoding these novel polyketide synthases are shown in SEQ ID NO: 33-
Listed as SEQ ID NO: 44 and SEQ ID NO: 115-SEQ ID NO: 120.

Another subject of the invention consists of a nucleic acid coding for a novel polyketide synthase I, characterized in that it comprises one of the nucleotide sequences SEQ ID NO: 34 to SEQ ID NO: 44 and SEQ ID NO: 115 to SEQ ID NO: 120. .

[0286]   Preferably, such nucleic acids are in isolated and / or purified form.

The present invention also includes SEQ ID NO: 34 to SEQ ID NO: 44 and SEQ ID NO: 115.
A recombinant vector comprising a polynucleotide comprising one of SEQ ID NO: 120.

The present invention also relates to a recombinant host cell comprising a nucleic acid selected from a polynucleotide comprising one of the nucleotide sequences SEQ ID NO: 34 to SEQ ID NO: 44 and SEQ ID NO: 115 to SEQ ID NO: 120, and the nucleotide sequences SEQ ID NO: 34 to It relates to a recombinant host cell comprising a recombinant vector into which a polynucleotide comprising one of SEQ ID NO: 44 and SEQ ID NO: 115 to SEQ ID NO: 120 has been inserted.

Preferably, the recombinant vector containing the DNA insert encoding the novel type I polyketide synthase of the present invention is a cloning and expression vector.

[0290]   Preferably, said recombinant host cell is a bacterium, yeast or filamentous fungus.

The amino acid sequences of novel polyketide synthases from organisms contained in soil samples have the nucleotide sequences SEQ ID NO: 34 to SEQ ID NO: 44 and SEQ ID NO: 11.
5 was estimated from SEQ ID NO: 120. They are polypeptides comprising one of the amino acid sequences SEQ ID NO: 48 to SEQ ID NO: 59 and SEQ ID NO: 121 to SEQ ID NO: 126.

The present invention also relates to novel polyketide synthases comprising an amino acid sequence selected from SEQ ID NO: 48 to SEQ ID NO: 59 and SEQ ID NO: 121 to SEQ ID NO: 126.

6 encoding the polypeptides of SEQ ID NO: 121 to SEQ ID NO: 126, respectively
The nucleotide sequence SEQ ID NO: 114, which also contains open reading frames,
It forms part of the invention.

The nucleotide sequence SEQ ID NO: 113 of the a26G1 cosmid containing a sequence complementary to SEQ ID NO: 114 also forms part of the invention.

In addition, Streptomyces co-
Elicolor) (ATCC No. 101.478), Streptomyces ambofaciens (N)
RRL number 2.420), Streptomyces lactamandurans (Str
eptomyces lactamandrans) (ATCC number 27.3)
82), Streptomyces rimo
sus (ATCC No. 109.610), Bacillus subtilis (Bacill)
us subtilis (ATCC No. 6633) or Bacillus lichenifornis and Saccharopolyspora erythrea.
Genomic DNA from a pure bacterial strain such as) was extracted and amplified according to the invention.

PCR amplification of DNA from each of the above bacterial strains was performed using a pair of primers specific for the nucleic acid sequence of type I polyketide synthase.

In this way, a novel bacterial type I polyketide synthase gene could be isolated and characterized. These are the nucleic acid sequences SEQ ID NO: 30 to SEQ ID NO: 32.

The subject of the present invention therefore also relates to a nucleotide sequence coding for a novel type I polyketide synthase selected from a polynucleotide comprising one of the nucleotide sequences SEQ ID NO 30 to SEQ ID NO 32.

Recombinant vectors containing a nucleotide sequence encoding the novel type I polyketide synthase described above form part of the invention.

The present invention also comprises a recombinant host cell comprising a nucleic acid encoding a novel type I polyketide synthase containing a nucleotide sequence selected from SEQ ID NO: 30 to SEQ ID NO: 32, and the above recombinant vector. And a recombinant host cell containing

A subject of the invention is also a polypeptide encoded by the sequence comprising nucleic acid SEQ ID NOs: 30-32, more particularly a polypeptide comprising the amino acid sequence SEQ ID NO: 47-SEQ ID NO: 50.

A subject of the invention is also: A nucleic acid encoding a type I polyketide synthase comprising a nucleotide sequence selected from SEQ ID NO: 33 to SEQ ID NO: 44 and SEQ ID NO: 30 to SEQ ID NO: 32 and SEQ ID NO: 115 to SEQ ID NO: 120. Producing a recombinant host cell containing: a step of culturing the recombinant host cell in an appropriate medium; recovering the type I polyketide synthase from the culture supernatant or the cell lysate; Is a method for producing a type I polyketide synthase of the present invention, which comprises a step of purifying.

The novel type I polyketide synthases obtained by the above method can be characterized by binding to an immunoaffinity chromatography column on which antibodies recognizing these polyketide synthases have been immobilized beforehand.

The Type I polyketide synthases of the present invention, and in particular the recombinant polyketide synthases described above, may also be expressed, for example, by reverse phase or anion exchange or cation exchange chromatography techniques well known to those skilled in the art. Can be purified by various high performance liquid chromatography (HPLC) techniques.

The recombinant or non-recombinant polyketide synthase of the present invention can be used for the production of antibodies.

In another aspect, the subject of the invention is also an antibody which specifically recognizes a type I polyketide synthase of the invention or a peptide fragment of such a polyketide synthase.

The antibodies of the invention can be monoclonal or polyclonal. The monoclonal antibody was obtained from Kohler and Milstein C. et al. (1975), Na
pure, Vol. It can be produced from hybridoma cells according to the technique described in 256: 495.

The polyclonal antibody may be an immunoadjuvant compound (eg
Manufacture by immunizing a mammal, in particular a mouse, rat or rabbit with a type I polyketide synthase of the present invention in the presence of complete Freund's adjuvant, incomplete Freund's adjuvant, aluminum hydroxide or compounds from the Muramyl peptide family. You can

For the purposes of the present invention, antibody fragments such as Fab, Fab ′, F (
ab ') ₂ or Martineau et al. (1998) J. Am. Mol. Biol.
, Vol. 280 (1): 117-127 or U.S. Pat. No. 4,946,77.
Single chain antibody fragment (ScFv) containing the variable portion described in No. 8, and Reinmann KA et al. (1997), AIDS Res. Hum. Retr
oviruses, Vol. 13 (11): 933-943 or Leger
O. J et al. (1997), Hum. Antibodies, Vol. 8 (1):
The humanized antibody described in 3-16 is also included in the "antibody".

The antibody preparations according to the invention may in particular detect the presence of a type I polyketide synthase according to the invention or for example this polyketide synthase in a cell lysate or culture supernatant of a bacterial strain capable of producing such an enzyme. Is useful in qualitative or quantitative immunological tests that are merely intended to quantify the amount of

Another subject of the invention is a method for detecting a type I polyketide synthase of the invention or a peptide fragment of this enzyme in a sample, comprising: a) the antibody of the invention as a test sample. Contacting, b) detecting potentially formed antigen / antibody complexes.

The invention also comprises the invention in a sample comprising: a) an antibody of the invention; b) where appropriate, the reagents necessary to detect possible antigen / antibody complexes formed. And a kit or device for detecting type I polyketide synthase.

Antibodies to the type I polyketide synthases of the invention can be labeled using detectable isotopic or non-isotopic labels according to methods well known to those of skill in the art.

A pair of primers that hybridize to a target sequence that is desired to be present, such as a sequence of the puromycin biosynthetic pathway, a sequence of the linA gene involved in lindane biodegradation, or a sequence encoding a type I polyketide synthase, is provided. The screening of the DNA libraries of the invention used is described in detail above.

The subject of the invention is therefore for the detection of a nucleic acid of a given nucleotide sequence or of a nucleotide sequence structurally similar to a given nucleotide sequence in a population of recombinant host cells of the invention. The method of: -recombining a population of recombinant host cells with a pair of primers that hybridize to a given nucleotide sequence, or a pair of primers that hybridize to a nucleotide sequence that is structurally similar to a given nucleotide sequence. A step of contacting; a step of performing at least three amplification cycles; a step of detecting the amplified nucleic acid;

Regarding amplification conditions suitable as a function of the desired target sequence, the person skilled in the art can advantageously refer to the examples below.

In another aspect, the invention also provides a nucleic acid of a given nucleotide sequence, or a nucleic acid of nucleotide sequence structurally similar to a given nucleotide sequence, in a population of recombinant host cells of the invention. A method for detecting, wherein: a population of recombinant host cells is hybridized to a given nucleotide sequence, or to a nucleotide sequence that is structurally similar to the given nucleotide sequence. Contacting; detecting a hybrid that may form between the probe and a nucleic acid contained within the vector of the population.

In order to screen the DNA libraries of the invention to detect the presence of nucleotide sequences that encode a polypeptide capable of degrading lindane, recombinant clones of interest are tested for their ability to degrade lindane. Were detected based on their phenotypes corresponding to. For this purpose, isolated clones and / or sets of clones of the prepared DNA library are cultivated in medium in the presence of lindane and the lindane degradation is carried out by immunizing the cells. ) Observed by the formation of a cloudy halo in the environment.

The present invention is also a method for identifying the production of a compound of interest by one or more recombinant host cells within a population of recombinant host cells of the invention, the recombinant host of said population. Culturing the cells in a suitable medium, detecting the compound of interest in the culture supernatant or cell lysate of the cultured one or more recombinant cells.

A subject of the present invention is also a method for selecting in a population of recombinant host cells according to the invention a recombinant host cell which produces a compound of interest, the recombinant host cells of said population. Culturing in a suitable medium, detecting the compound of interest in the culture supernatant or cell lysate of one or more cultured recombinant host cells, recombinant host cell producing the compound of interest The method is characterized by including the step of selecting.

The present invention also includes the step of culturing the recombinant host cell selected by the above method, recovering the compound produced by said recombinant host cell and, if appropriate, purifying And a method for producing a compound of interest.

The present invention also relates to the compounds of interest, characterized in that they are obtained by said method.

The compounds of interest of the present invention express at least one nucleotide sequence comprising a sequence selected from SEQ ID NO: 33 to SEQ ID NO: 44 and SEQ ID NO: 30 to SEQ ID NO: 32 and SEQ ID NO: 115 to SEQ ID NO: 120. It may consist of a polyketide produced thereby.

The present invention is also prepared by expressing at least one nucleotide sequence comprising a sequence selected from SEQ ID NO: 33 to SEQ ID NO: 44 and SEQ ID NO: 30 to SEQ ID NO: 32 and SEQ ID NO: 115 to SEQ ID NO: 120. And a composition comprising a polyketide.

The polyketides produced by expressing said at least one nucleotide sequence are preferably several coding sequences contained within a functional operon whose translation products are the various enzymes required for polyketide synthesis. (One of the sequences is contained within and expressed in the operon) is the product of activity. Such operons containing a nucleic acid sequence of the invention encoding a polyketide synthase can be constructed, for example, according to the teaching of Borchert et al. (1992).

The present invention also relates to pharmaceutical compositions comprising a pharmacologically active amount of a polyketide of the invention and, where appropriate, a pharmaceutically acceptable vehicle.

Such a pharmaceutical composition may be 1 μg / kg / day to 10 mg / kg / day, preferably at least 0.1, synthesized by a type I polyketide synthase according to the present invention.
It will advantageously be applied to the administration of polyketides in an amount of 01 mg / kg / day, most preferably 0.01 to 1 mg / kg / day, eg parenteral administration.

The pharmaceutical composition of the present invention can be administered orally, rectally, parenterally, intravenously, subcutaneously or intradermally.

The invention also relates to the use of a polyketide obtained by expressing a type I polyketide synthase according to the invention for the manufacture of a medicament, in particular a medicament having antibiotic activity.

The invention is also illustrated by the figures and examples below, which do not limit the invention in any way.

FIG. 1 shows a scheme of various cell lysis steps performed according to the protocols 1, 2, 3n, 4a, 4b, 5a and 5b described in Example 1.

FIG. 2 shows that 0 mg of DNA extracted from 300 mg of soil No. 3 (St Andre coast) after various cell lysis treatments (protocols 1-5, see FIG. 1).
． Electrophoresis on an 8% agarose gel is shown. M: Lambda phage molecular weight marker.

FIG. 3 shows the proportions of various genera of actinomycetes cultured after treatments 1-5 (see FIG. 1). Cfu (colony forming units) values were measured on media selective for this group of bacteria. A total of about 400 colonies were analyzed.

FIG. 4 shows Hin added to soil at various concentrations before (G) or after (G ^* ) milling.
6 shows recovery of lambda phage DNA digested with dIII. Treatment T (heat shock)
And S (sonication) are additional cell lysis treatments. The quantification was performed by analysis on a phosphoimager after dot blot hybridization. Samples of each soil were used for each concentration of lambda phage added. The characteristics of the soil are shown in Table 1. Samples corresponding to the added 10-15 μg pf DNA were untreated.

FIG. 5 shows PCR amplification of DNA extracted from soil number 3 according to protocols 1, 2, 3, 5a and 5b. Resident Streptosporangium species (Strep
tosporangium spp. ) For targeting
S 122 and FGPS 350 (Table 2) were used. The extracted DNA was used undiluted or at 10-fold and 100-fold dilutions. M: 123 bp molecular weight marker (Gibco BRL), C: amplification control without DNA.

[0336] Figure 6 shows S. lividans OS48.
． DNA extracted after inoculating the soil with various concentrations of spores or mycelium of No. 3
Indicates the amount of The amount of mycelium added to the soil corresponds to the number of spores inoculated into the germination medium. About 50% of the spores germinated and the number of cells or genome contained in the germinated spore hyphae was not determined. Therefore, the amount of inoculated spores and mycelia cannot be directly compared. The extraction protocol was performed according to Protocol 6 (see Materials and Methods section). The symbol (') indicates the RN in the extraction buffer.
It indicates that A was included. The target DNA was amplified by PCR with primers FGPS 516 and FGPS 517 and the quantification was performed using the probe FGPS 5
Performed on a phosphoimager after dot blot hybridization using 18. Samples of each soil were used for each concentration of hyphae or spores. The characteristics of the soil are shown in Table 1.

FIG. 7 represents a phylogenetic tree obtained with the Neighbour Joining algorithm which positions the 16S rDNA sequences contained in the soil DNA library relative to the culture reference bacteria. Gray display: sequences obtained from a pool of clones of the library.

The bootstrap values after 100 iterations of resampling are shown at the aggregation points. The scale line shows the number of substitutions per site. The access numbers for the sequences in the Genbank database are shown in parentheses.

[0339]   FIG. 8 represents the scheme of the vector pOSint1.

[0340]   FIG. 9 represents the scheme of the vector pWED1.

FIG. 10 represents the scheme of the vector pWE15 (ATCC No. 37503).

[0342]   FIG. 11 represents the scheme of the vector pOS700I.

[0343]   FIG. 12 represents the scheme of the vector pOSV010.

FIG. 13 represents the fragment containing the “cos” site inserted into plasmid pOSV010 during the construction of vector pOSV303.

[0345]   FIG. 14 represents the scheme of the vector pOSV303.

[0346]   FIG. 15 represents the scheme of the vector pE116.

[0347]   FIG. 16 represents the scheme of the vector pOS700R.

[0348]   FIG. 17 represents the scheme of the vector pOSV001.

[0349]   FIG. 18 represents the scheme of the vector pOSV002.

[0350]   FIG. 19 represents the scheme of the vector pOSV014.

[0351]   FIG. 20 represents the scheme of the vector pBAC11.

[0352]   FIG. 21 represents the scheme of the vector pOSV403.

FIG. 22 represents an electrophoretic gel of the DNA of the library after digesting positive clones of the library screened with PKS-1 oligonucleotides with the enzymes BamHI and DraI.

[0354] Figure 23 shows the production of puromycin by the S. lividans recombinant as compared to the production of the S. alboniger wild type strain.

FIG. 24 shows an alignment of soil PKS with the conserved active site of other PKSs. The display content for each peptide is shown. The beta-ketoacyl synthase domain has the GCG PILEUP program (Wisconsin P
ackage Version 9.1, Genetics Computer
Aligned using Group, Madison, Wisc).

[0356]   FIG. 25 shows the construction of an integrative mating cosmid.

[0357]   FIG. 26 shows the construction of a built-in mating BAC.

[0358]   FIG. 27 shows a scheme for the construction of vector pOSV308.

[0359]   FIG. 28 shows a scheme for the construction of vector pOSV306.

[0360]   Figure 29 shows the scheme for the vector pOSV307.

[0361]   FIG. 30 shows a scheme for the construction of vector PMBD-1.

FIG. 31. Detailed map of plasmid pMBD-2 and vector pMBD-3.
A scheme for the construction of is shown.

[0363]   Figure 32 shows a detailed map of plasmid pMBD-4.

FIG. 33 shows a scheme for the construction of plasmid pMBD-5 from plasmid pMBD-1.

[0365]   Figure 34 shows a detailed map of the vector pBTP-3.

FIG. 35 shows a scheme for the construction of vector pMBD-6 from vector pMBD-1.

FIG. 36 shows a map of cosmid a26G1 whose DNA insert contains an open reading frame encoding several polyketide synthases.

FIG. 37 is a scheme depicting the DNA insert (+ strand) of cosmid a26G1 arranged with various reading frames encoding several polyketide synthases.

[0369]     (Example)   Example 1: Containing organisms, including the step of direct extraction of DNA from soil samples For producing nucleic acid population from soil sample   1. Materials and methods   1.1soil: The characteristics of the 6 soils used in this study are shown in Table 1.

The range of clay content and organic matter content is 9-47% and 1.7-, respectively.
4.7%, pH range 4.3-5.8.

Soil samples were collected from the surface 5-10 cm deep. Remove all visible roots and optionally store the soil at 4 ° C for several days, then dry them at room temperature for 24 hours, sieve (average mesh size: 2mm), then at 4 ° C. Stored for up to several months.

[0372]   1.2 Bacterial strain and culture conditions   Supply vegetative cells, spores or mycelia used to inoculate the soil sample
Bacterial strains and extracellular DNA so that their presence can be closely monitored.
Selected.

To obtain large amounts of extracellular DNA, a lysogenic strain of E. coli 1192 Hfr P4X (metB) containing lambda phage CI857 Sam7 was added at 30 ° C. on Luria-Bertani (LB) medium. 2 hours, then 40
The culture was carried out at 30 ° C. for 30 minutes and then at 37 ° C. for 3 hours. The lambda phage DNA was replaced with S
ambrook J. Et al. (1989) Molecular Cloning: A
Laboratory Manual, Second Edition, Cold Spring H
Arbor Laboratory, Cold Spring Harbor
N. Y. It was extracted according to the technique described in 1.

An Abilent Strain of Bacillus anthracis (STE)
RNE 7700) was used as a bacterial cell inoculum. Bacillus
s anthracis) "Tripty Case Soy Broth" (TBS) (B
iomerieux, Lyons, France) for about 6 hours,
It was confirmed that the OD ₆₀₀ was maintained below 0.6. These conditions allow vegetative cell growth without spore formation (Patra et al., (1996).
, FEMS Immunol. Medical Microbiology, v
ol. 15: 223-231). Streptomyces lividans (Strep
tomyces lividans) OS 48.3 (Clerc-Bardin)
Spores of R2YE medium (Hopwood et al., (1985), Ge.
net Manipulation of Streptomyces-A
Laboratory Manual. The John Inns Fo
were mechanically removed from the biological culture on the United Nations, Norwich, United Kingdom). Streptomyces lividans
dans) OS48.3 hyphae were obtained from pregerminating spores. This is because the use of short hyphae was expected to minimize DNA destruction and subsequent loss. The spores were treated with TES buffer (N-tris [hydroxymethyl] methyl-2.
-Aminoethane-sulfonic acid; Sigma-Aldrich Chimie, F
rance (0.05 M; pH 8) (Holben WE et al., (1988), A.
PPL. Environ, Microbiol. vol. 54: 703-711
), Followed by heat shock (10 minutes at 50 ° C., then cooling under cold running water, and then an equal volume of pre-germination medium (1% yeast extract, 1% casamino acid, 0.
01M CaCl ₂ )).

The solution was incubated at 37 ° C. on a stirrer. Hopwood et al. (19
As a result of 85), the ratio of germinated spores was estimated to be about 50%. After centrifugation, the pellet was resuspended in TES buffer and added to 3% TSB medium,
． Incubated at 37 ° C. until an OD _{450 of} 15 was obtained (Hopwoo
d et al. (1985)). Streptomyces hygroscopicus (Strep
tomyces hygroscopicus SWN 736 and Streptosporangium flag
ile) AC1296 (Institution Pushino, Moscow)
Were cultured according to the technique described by Hickkey and Tresner (1952).

DNA of S. lividans spores and hyphae was extracted from pure cultures according to the Cell Lysis Protocol 6 below (but without grinding), while Streptomyces hygrostis was used. Copics (S
． hygroscopicus) and Streptosporangium flazil (
S. Fragile spores are chemically / enzymatically lysed (Hinterman).
n et al., 1981).

[0377]   1.3 Selection of extraction buffer   TENP buffer (50 mM developed by Picard (1992)
Tris, 20 mM EDTA, 100 mM NaCl, 1% wt / vol
Polyvinyl polypyrrolidone) was used. Later, similar buffers were created by other authors (
Cleggg et al., 1997; Kusk et al., 1998; Zhou et al., 1996).
Used more.

The Tris and EDTA protect DNA from nuclease activity and
1 produces a dispersing effect, and the PVPP absorbs humic acid and other phenolic compounds (Holben et al., (1988); Picard et al., (1992).
)).

In this study, the effectiveness of extraction of this buffer was evaluated at a p of 5.8-8.3.
Twenty different soils with H range and organic matter content of 0.2-6.3% were used and evaluated at different pH values (6.0-10.0). These 20 soils (
Other features not shown) were used only in this experiment. The amount of DNA is
It was measured by the colorimetric means described in Richard (1974) and described in detail below.

[0380]   1.4 Protocol for in situ cell lysis and DNA extraction   Evaluate the effectiveness of various techniques for cell lysis of soil microorganisms in situ
In order to do so, several protocols were tested with increasing numbers of steps. this
In these experiments, the indigenous soil microbiota was targeted in 6 soils. To the soil
The amount and quality of the recovered DNA derived from the added lambda phage DNA.
Study the effect of the cytolytic treatment on free DNA by analyzing
Therefore, additional experiments were performed.

Once an optimized protocol (designated Protocol 6) was developed,
This protocol was used to quantify DNA from indigenous actinomycetes and DNA from Gram-positive bacteria inoculated into selected soil. In all cases,
The soil sample was dried and sieved as above.

After milling, 0.5 ml TENP buffer was added to 200 mg dry weight of soil. However, in Protocol 1, the buffer was added to unground soil.

For various cell lysis treatments (see below), the soil suspension was vortexed for 10 minutes, centrifuged (4000 × g for 5 minutes), then an aliquot of the supernatant. Minutes (25 μl) were analyzed by gel electrophoresis (0.8% agarose).

Another aliquot fraction of the supernatant, corresponding to a known volume (generally 350 μl), was precipitated with isopropanol.

Five aliquot fractions (representative of DNA from 1 g soil) were combined and 100 μl sterile TE buffer (10 mM Tris, 1 mM EDTA).
, PH 8.0) and then purified (Protocol D, see below) and quantified. This was done by hybridization of the total DNA (Dot-Blot) or by hybridization of the PCR amplification product (dot blot) (see below).

The hybridization signal was quantified by phosphorescence imaging (“phosphoimaging” technique, see below).

[0387]   1.5 Evaluation of methods for in situ cell lysis   Extracted after an increasing number of cell lysis steps (protocols 2-5b)
The quality and quantity of DNA was determined by the extracellular D obtained after washing the soil with extraction buffer.
Compared to the case of NA (Protocol 1; see also Figure 1).

[0388]   Protocol 1: No cell lysis treatment   Add TENP buffer to unmilled soil and perform the DNA extraction process as described above.
It was.

[0389]   Protocol 2: Soil crushing and subsequent DNA extraction   The soil was ground using two different types of equipment.

To compare the effectiveness of each of them, 5 g of dry soil were crushed in a crusher containing a tungsten ring for 30 seconds or 4 times varying up to 60 minutes, mortar and agate beads ( Milled in a soil mill containing 20 mm diameter).

[0391]   The TENP buffer was then added and the DNA was extracted as above.

The results of the gel electrophoresis show that to obtain an amount of extracted DNA equivalent to that obtained after 30 minutes of milling using a tungsten ring, agate beads are used.
It was shown that grinding for a minute was required.

[0393]   The size distribution of the DNA fragment is the same regardless of which method is used.

Therefore, these treatments are considered equivalent and, therefore, the treatments used in the protocols below are not specified.

In Protocols 3-5, the effectiveness of several other cell lysing treatments after milling the soil was tested independently or in various combinations.

[0396]   Protocol 3   This protocol is the same as Protocol 2. However, it takes 5 minutes
Ultra-turrax type mixer (Ja
nker and Kunkel, IKA Labortechnik, Germany
homogenization step using ny).

[0397]   Protocols 4a and 4b   These protocols are the same as Protocol 3 except for an additional sonication step.
It is the same.

Two types of sonicator devices, a titanium micropoint sonicator (600W Vibracell Ultrasonicator, Bio.
block, Illkirch, France) (protocol 4a) and C
The up Horn type sonicator (protocol 4b) was compared.

Vibracell micropoints that generate ultrasound are in direct contact with the soil solution.

For the Cup Horn type device, the soil solution is supplied in tubes placed in a water bath that mediates the passage of ultrasonic waves.

Preliminary experiments were performed to determine the optimal conditions for those two sonicators (results not shown).

The best compromise in terms of the amount of DNA extracted and the size of the fragments was to adjust the power to 15W with a 50% active cycle for 7 minutes and 10 minutes on a Titanium Micropoint and Cup Horn type sonicator respectively. Sonication.

[0403]   Protocols 5a and 5b   Ultrasonic treatment with Titanium Micropoint or Cup Horn type equipment (
After protocols 4a and 4b), respectively, lysozyme and achromopep
Tidase was added to a final concentration of 0.3 mg / ml of each of the enzymes.
.   The soil suspension was incubated at 37 ° C for 30 minutes and then a final concentration of 1% lath.
Urylsulfate was added, then the suspension was incubated at 60 ° C for 1 hour.
Then, it was centrifuged and precipitated as described above.

In addition to the protocol described above, sonication (see Cup Horn, protocol 4b) on HindIII-digested lambda phage DNA previously added to the soil and heat shock (30 seconds in liquid nitrogen, The effect of these treatments was then repeated 3 times in boiling water for 3 minutes) (see below).

In the prior art, heat shock has been suggested as a means for in situ cell lysis (Picard et al. (1992)). However, such treatment was not included in the above protocol, as it had a deleterious effect on the free DNA (see Results section).

Optimized Protocol The optimized protocol was determined after evaluation of the various cell lysis treatments. It is referred to as Protocol 6 . Protocol 6 is the same as Protocol 5b except that the soil suspension was vortexed prior to sonication, then stirred by spinning on a turntable for 2 hours and then frozen at -20 ° C.

After thawing, the soil suspension was vortexed for 10 minutes before sonication. Protocol 6 was used in experiments inoculating the soil with bacterial cells and in experiments quantifying indigenous actinomycetes (see below).

[0408]   1.6 Microscopic counting   The effectiveness of grinding the soil as a method for lysing bacterial cells was examined by microscopy.
Investigated by.

5 g of dry crude soil was mixed with 50 ml of sterile ultrapure water in a Waring Blender apparatus for 1.5 minutes, while at the same time 1 g (dry weight) of ground soil (protocol 2) was stirred for 10 minutes. Suspended in 10 ml. The soil suspension was serially diluted and acridine orange was added to a final concentration of 0.001%.

After 2 minutes, the suspension was filtered through a 0.2 μm black type Nucleopore brand membrane. Rinse each filter with sterile water with cells lysed
The bacteria were fixed by treatment with 1 liter of isopropanol for 1 minute and then rinsed again.

The bacterial cells were transferred to Zeiss Universa equipped with a 100 × objective lens.
1 Epifluorescence microscopy was used to count. For each soil type, 3 filters were counted and at least 200 cells were counted on each of those filters.

[0412]   1.7 Count of culturable actinomycetes and total number of colony forming units (CFU)   Actinomycetes that survived the cell lysis treatment (protocols 1 to 5) were treated with soil numbers.
3 (See Saint Andre Coast, see Table 1).

SDS (0.05) to induce germination (Hayakawa et al. (1988)).
%) And yeast extract (6% w / v), after a 10-fold dilution, the soil suspension was serially diluted in sterile water and incubated at 40 ° C. for 20 minutes to obtain HV medium (
Hayakawa et al. (1987)).

The HV medium was treated with actidione (50 mg / l) and nystatin (50 mg / l).
l) was supplemented.

[0415]   After incubation for 15 days at 28 ° C., the actinomycete colonies were counted.

A total of about 400 colonies were examined. The identification was based on gross and microscopic morphological features and on analysis of the diaminopimelic acid content of the isolate (Shirring et al., 1966); Staneck et al., 1974.
Williams et al., 1993).

The total amount of culturable bacteria (total CFU) was also measured for each of the cell lysis protocols 1-5. The soil suspension was serially diluted and Bennett agar medium (W / W) supplemented with nystatin and actidione (50 mg / l each).
Aksman et al., 1961) were inoculated in triplicate.

Cover each Petri dish with a cellulose nitrate filter (Millipore) and
Incubated at C for 3 days. After counting the colonies on the membrane, the filter was removed, the Petri dishes were reincubated at 28 ° C. for 7 days and then counted again.

[0419]   1.8 Recovery of lambda phage DNA added to soil   The lambda duff was prepared according to standard protocols (Sambrook et al., 1989).
The digested DNA was digested with HindIII and extracted with a phenol-chloroform mixture.
It was taken out, allowed to settle and then resuspended in sterile ultrapure water.

DNA 0, 2.5, 5, 7.5, 10 and 1 per gram dry weight of soil
Dilute each dilution to 5 μg (μg DNA / g soil dry weight)
Prepared in a volume of l. Add these DNA dilutions to a 5 g batch of dry soil,
It was then vortexed vigorously for 5 minutes before grinding.

Before crushing, the lambda phage DNA was treated with 0, 10 and 15 μg DN.
A / g soil dry weight was added to the soil.

After milling, the extraction buffer was added and the DNA was extracted according to Protocol 2 (see above).

[0423]   1.9 Saturation of adsorption sites with RNA   Saturation of nucleic acid adsorption sites of the soil colloid can increase recovery levels of the DNA
To determine whether or not sandy soil (soil no. 4) and clay before other treatments.
Soil soil (Soil No. 5) was incubated with the RNA solution.

Commercially available Saccharomyces cerevisiae
visiae) RNA (Boehringer Mannheim, Meyla
n, France) was diluted in phosphate buffer (pH 7.1) and dried at 20, 50 and 100 mg R in dried soil samples (2 ml / g soil).
The final concentration of NA / g soil dry weight was added.

The tube containing the soil suspension was agitated by rotation for 2 hours at room temperature. After centrifugation, the soil pellet was dried in an oven (50 ° C) overnight. The Lambda phage DNA (0, 20 or 50 μg / g soil dry weight) was then added to the soil to mimic the fate of DNA released after cell lysis.

The DNA was extracted according to Protocol 2. Then R for DNA recovery
It was confirmed that the same effect of adding NA can be achieved by adding the RNA directly to the extraction buffer.

This simplified method was used for clayey soil No. 5 in the experiment of inoculating the soil with the microorganism.

The RNA was then added at a concentration corresponding to 50 mg RNA / g soil dry weight.

[0429]   1.10 Qualitative and quantitative determination of the effectiveness of the extraction protocol   The quality of the DNA (in the absence of degradation) was determined by lysis on a 0.8% agarose gel.
Relative position of DNA migration band or DNA breakage after electrophoresis of aliquot fraction of liquid
It was evaluated based on the size of the piece.

[0430]   The fluorescence intensity allowed a semi-quantitative estimation of the extraction yield.

Another aliquot fraction was used for quantitative determination of DNA content by hybridization (dot blot) and analysis on a phosphoimager. The dot blot (dot blot) hybridization protocol is Si
described by monet et al. (1990).

The hybridization membrane (GeneScreen Plus, Life Sc
Ience Products, Boston, USA), 6 ml of 20 × S
Prehybridized in SC, 1 ml Denhardt's solution, 1 ml 10% SDS and 5 mg salmon sperm DNA in 20 ml solution for at least 2 hours.

The hybridization was carried out overnight in the same solution in the presence of labeled probe, followed by two washes of the membrane in SSC 2 × buffer for 5 minutes at room temperature,
Then, a third wash in SSC 2x, 0.1% SDS buffer and a fourth wash in SSC 1x, 0.1% SDS buffer for 30 minutes at the hybridization temperature were performed.

The hybridization signal was analyzed by the Biorad radioactivity imaging system (Molecular Analyst Software, BIORAD, I.
Vry-sur-Seine, France).

To quantify the total amount of DNA from indigenous microflora, protocols 1-5
Various soils were extracted according to. The unamplified DNA was applied to a dot blot membrane and hybridized using the universal probe FGPS431 (Table 2).

E. coli 16S rDNA gene (Amann et al. (1995)
) At position 1392-1406 of polynucleotide T4 kinase (Boehringer Mannheim, Melan, F.).
labeled at the end with ³² P ATPα.

A calibration curve was generated using E. coli DH5α DNA. The conversion of the calculation to the soil bacteria required a simplification starting from the assumption that the average number of copies (rrn) for E. coli was 7.

The extracellular DN was isolated using HindIII digested lambda phage DNA.
The recovery of A was quantified. An unamplified extract from soil supplemented with lambda phage DNA was digested with HindIII and the Klenow fragment (Boehringer).
Mannheim, Melan, France) were used to hybridize to randomly labeled lambda phage DNA.

The amount of DNA was determined by an interpolation method using a calibration curve prepared with the purified DNA.

The total amount of DNA extracted from soils 1, 2, 3, 4 and 6 according to Protocol 2 (grinding) was also quantified by colorimetric means according to the technique described by Richard (1974).

Briefly, the DNA was converted to concentrated HClO ₄ (final concentration of HClO ₄ was 1.5
Was N). 2.5 volumes of this solution were mixed with 1.5 volumes of DPA (diphenylamine, Sigma-Aldrich, France), the mixture was incubated at room temperature for 18 hours and then the OD at 600 nm was measured. The soil DNA extract was subjected to standard protocols (Sambrook et al., (1989).
) Was used to quantify against a standard curve prepared with DNA extracted from E. coli DH5α.

[0442]   1.11 DNA Quantification Technique Using PCR Amplification and Hybridization development of   For PCR amplification, DNA Taq polymerase (Appligene Onc
or France) was used according to the manufacturer's instructions.

The PCR program used for all of the amplifications is as follows: 3 at 95 ° C.
Initial denaturation in minutes, followed by 35 cycles consisting of 95 ° C for 1 minute, 55 ° C for 1 minute and 72 ° C for 1 minute, followed by a final extension at 72 ° C for 3 minutes.

Streptosporangium
DNA isolated and purified from Fragile) was used as a control at a concentration of 100 fg to 100 ng.

In order to specifically amplify the DNA of this bacterium, the primer FGPS complementary to a part of 16S rDNA after alignment of the sequence of Actinomyces 16S rDNA.
122 and FGPS350 (Table 2) were selected. Streptomyces strains (Streptomyces), Streptosporangium (Strepto)
sporangium) and other very similar genera) and their specificity was tested.

The PCR product was hybridized to the oligonucleotide probe FGPS643 (Table 2). To mimic the level of purity obtained with DNA extracted from the soil, a control of pure DNA from S. fragile was obtained after treatment according to cell lysis protocols 4b and 5b. And then mixed with soil extract purified according to Protocol D.

Prior to use, the soil extract was treated with DNase (1 unit of DN for 30 minutes at room temperature).
Ase / ml, Gibco BRL). Then, the DNase was heated to 65 ° C.
It was inactivated by heating for 10 minutes. Confirmation of the inactivation was performed by PCR. Humic acid concentration was measured by spectrophotometry (OD ₂₈₀ nm) against a standard curve of commercially available humic acid (Sigma).

Soil solutions treated with undiluted, 10-fold and 100-fold diluted DNase were
Prior to the PCR amplification, it was mixed with 100 fg to 100 ng of S. fragile DNA. In another series of experiments, to mimic the presence of non-target DNA and its effect on the PCR method,
Increasing concentration of Streptomyces hygroscopicus (Strept
omyces hygroscopicus) DNA (100 pg to 1 μg) was added to the S. fragile DNA.

[0449]   1.12 Purification of crude DNA extract   The four DNA purification methods were compared. 1 g of the DNA according to Protocol 4a
Extracted from (dry weight soil), 100 μl of buffer TE8 (50 mM T
ris, 20 mM EDTA, pH 8.0).

[0450]   Protocol A   Two consecutive Elutip d columns (Schleicher and Sc
huell, Dassel, Germany) (Picard et al., (1992).
) Via elution.

[0451]   Protocol B   Sephacryl S200 column (Pharmacia Biotech
, Uppsala, Sweden) and subsequent Elutip
  e Elution through a column (Nesme et al. (1995)).

[0452]   Protocol C   17.9% (w / w) PEG 8000 (Merck, Darmsta
dt, Germany) and 14.3% (wt / wt) of (NH_Four)_TwoSO_Four Separation using a two-phase aqueous system by (Zaslavsky et al., (1995)).

[0453]   After vigorous vortex mixing, the two phases were left to separate at room temperature.

1 ml of each of the phases was transferred into another tube, mixed with 100 μl of the sample and left to separate overnight at 4 ° C.

To remove excess salt, the lower phase was washed with excess TE7.5 buffer (10 m
Dialysis was performed through Millipore membrane for 1 hour in the presence of M Tris, 1 mM EDTA (pH 7.5) and 1 M MgCl ₂ .

[0456]   Protocol D   Microspin Sephacryl S400 HR column (Phar
elution via Macia Biotech, Uppsala, Sweden)
And subsequent elution through an Elutip d column.

Each protocol is completed by the step of precipitation with ethanol. The DNA is 1
Resuspend in 0 μl TE 7.5 buffer. PCR efficiency of the undiluted aliquot fractions of the DNA solution and 10-fold and 100-fold diluted aliquot fractions was tested using standard protocols (see below).
Confirmed by amplification.

[0458]   1.13 Recovery of DNA from inoculated microorganism   The cells, spores and hyphae were washed twice and by counting on a plate or directly
It was counted by various microscopic counts. A batch of 5 g of sieved dry soil (soil
Lom 2, 3 and 5), 0, 10^Three10,⁵10,⁷And 10⁹Spore / g soil
Concentrations corresponding to dry weight of Streptomyces lividans (S. livida
ns) spore and hypha suspension 100 μl, or 0, 10⁷And 10⁹Fine
B. anthracis nutrient at a concentration corresponding to dry cell / g soil dry weight
Cells were inoculated.

The amount of S. lividans hyphae was calculated based on the number of spores from which they were derived. After addition of the bacterial suspension, the soil sample was vortexed vigorously for 5 minutes and then ground. The DNA was extracted according to Protocol 6 (see below).

To quantify the amount of DNA recovered from the cells and spores and from the bacterial mycelium inoculated into the soil, PCR amplification followed by dot blot hybridization and phosphorescence imaging (phosphoimaging) was performed. )
Was used.

The DNA extraction was performed according to the cell lysis protocol 6. The PCR amplification and the hybridization were performed as described above. The primers and probes are targeted to chromosomal regions located outside the 16S region and are highly specific to each organism to avoid background signals.

For the soil inoculated with B. anthracis, primer R499
And R500 (Patra et al. (1996)) were used to hybridize the amplification product to the oligonucleotide probe C501 (Table 2).

For the soil inoculated with S. lividans, PCR reaction was performed using primers FGPS516 and FGPS517, and the amplified product was hybridized with the oligonucleotide probe FGPS518 (Table 2). .

The amplification region is part of a cassette specially constructed to obtain strain OS48.3 (Clerc-Bardin et al., Unpublished).

The calibration counts were in all cases obtained using purified DNA from the target organism.

[0466]   2. result   2.1 Selection of extraction buffer   20 different soils were used to determine the optimum pH of the DNA extraction buffer.
used. For all soils, the DNA yield increases with the buffer pH.
Increase as Calculated as a percentage of the highest value for each of the soils
The yield (± sd) for each pH issued is as follows: pH 6.0: 3.
1 ± 13; pH 7.0: 43 ± 16; pH 8.0: 60 ± 14; pH 9.0: 8
2 ± 12; pH 10.0: 98 ± 3.

In 16 out of 20 soils, the highest yield was obtained at pH 10.0, while in other soils the highest yield was obtained at pH 9.0. However, at pH 10.
At 0, more humic material was released than at pH 9.0 (results not shown). Therefore, pH 9.0 was selected in all of the experiments described below.

[0468]   2.2 Effectiveness of DNA extraction protocol   To evaluate the efficacy of several in situ cytolysis protocols,
Total DNA from indigenous soil microorganisms was extracted and quantified. Soil samples 1-6 (Table 1
) Was treated according to protocols 1-5 described in the Materials and Methods section (FIG. 1).
.

After the DNA extraction, the soil suspension was precipitated with isopropanol and an aliquot fraction of the resuspended pellet was analyzed in the first step by gel electrophoresis to determine the quality and quantity of released DNA. evaluated.

However, as the number of cell lysis steps increased, the color of the DNA extract became darker due to co-extraction of compounds such as humic acid with the DNA.

Some of these dark crude extracts do not migrate within the agarose gel in the expected manner.

Therefore, the crude DNA solution was purified (Protocol B) before quantification. Gel electrophoresis of the purified solutions obtained after various cell lysis treatments is shown as an example for soil 3 (Fig. 2).

A visual comparison of the intensity of the colored DNA by UV irradiation allowed a semi-quantitative assessment of the effectiveness of the treatment. In addition, DNA fragments of multiple sizes (discontinuous bands)
The presence of the migration (migration) profile and the disappearance of long fragments indicates that degradation of the DNA is occurring.

[0474]   DNA could not be extracted from the clay soil number 5.

A more precise quantification of DNA from their total soil extracted according to Protocols 1-5 was performed using an oligonucleotide probe (probe FGPS431, Table 2) complementary to the highly conserved sequences of the 16S rDNA region. ) And P in advance
Dot blot hybridization was performed without CR amplification.

The DNA was detected in extracts of all soils, except clayey soil number 5, after each of their various cell lysis steps.

[0477]   The results are in agreement with the evaluations made after gel electrophoresis.

For comparison with an independent quantification method, DNA extracted from Protocol 2 (from all soils except soil number 5) was analyzed by a colorimetric DNA detection method (Ri
chard, 1974).

A good correlation was found between DNA quantified using this colorimetric technique and the results obtained by dot blot hybridization / radio imaging (r = 0.88). This means that the average number of copies of the soil bacteria (rrn)
Confirms the assumption that is 7.

The hybridization (dot blot) showed that the amount of extracellular DNA measured by extraction without cell lysis treatment (protocol 1) was 4 μg / g for acidic soil (number 6) to 36 μg / g for soil number 3. It is shown that the range is up to (Table 3).

Milling the soil (Protocol 2) increased the amount of DNA extracted from all soils (eg 26 μg / g soil for soil number 6 and 59 μg / g soil for soil number 3) ( Table 3; FIG. 2).

For the two milling treatments (see Materials and Methods section), the discontinuous DNA migration was detected on the agarose gel, which indicated that the DNA molecule was partially degraded. Is shown (FIG. 2).

The size of the DNA fragment is 20 to 0.2 kb. The band intensity of the smallest fragment is very low, indicating that most of the fragment is much larger than 1 kb.

Protocol 3 refers to Ult after adding the extraction buffer to the soil sample.
Including a step of homogenization in a ra-turrax mixer. This step resulted in an increase in the amount of extracted DNA as measured by dot blot hybridization on two of those soils (sandy soil number 3 and acidic soil number 6),
On the other hand, two soils rich in organic matter (Soil No. 1 and No. 2) have less D
Resulted in the production of NA.

Protocols 4a and 4b are DNA from pre-milled and pre-homogenized soil
It was possible to evaluate the effect of the two types of sonication on the yield of.

The sonication had no positive effect on the DNA yield, except for soil number 6, as compared to protocol 3. However, the effectiveness of cell lysis for those two types of sonicator is different. For soils 2, 3 and 4, the highest amount of extracted DNA was obtained when using titanium micropoints (Table 3; Figure 2), while for soil numbers 1 and 6 the Cup Horn device was used. In some cases, the DNA yield was higher.

When an enzymatic / chemical cell lysis step is added after the sonication step (
Protocols 5a and 5b), conflicting results were obtained. In some cases the amount of DNA extracted was higher than that recovered according to protocols 4a and 4b, while in other cases the yield was lower (Table 3).

[0488]   2.3 Direct counting of microorganisms   Mill microscopic counts of total number of bacterial cells after staining with acridine orange
Was performed on all soils before and after.

Before crushing, the number of bacteria per gram of dry weight of soil was 1.4 × 10 ⁹ (± 0.4) in tropical soil number 5, from soil (Soil number) obtained from the Saint-Andre coast. The range was up to 10 × 10 ⁹ (± 0.7) in 3) (Table 1).

After crushing, the cell numbers were 45, 74, respectively, which were the initial values for soil numbers 1-6.
, 75, 54, 34 and 75%.

[0491]   2.4 Count of culturable actinomycetes belonging to various genera   Modifications in the actinomycete population in soil number were not recognized after various cell lysis treatments.
(Fig. 3).

For example, the colonies of Streptomyces sp. Dominate the viable Actinobacterial flora when the lysis treatment is not applied (Protocol 1) and the total number of identified colonies. Equivalent to 65%. After crushing
The proportion of Streptomyces colonies dropped to 51%, while the proportion of colonies belonging to the genus Micromonospora increased by 14% to 41%.

The chemo / enzymatic cell lysis (protocols 5a and 5b) appeared to be particularly effective in lysing Streptomycetes. When all cell lysis treatments including chemical / enzymatic cell lysis (protocols 5a and 5b) were applied, the actinomycete microflora still containing more than 10 ⁶ cfu / g soil was Micromonospora. It was dominated by species belonging to the genus, while little or no Streptomyces colonies were recovered.

Streptosporangium, Actinomadura, Microbispora (Microbis)
Pora), dactylosporangium
And organisms belonging to genera such as Actinoplanes were found in minor numbers on the plate after grinding, homogenization on an Ultra-turrax device and sonication (2 of the total number of identified colonies). ~ 8%), and was largely absent when these treatments were combined with chemical / enzymatic cell lysis.

In addition, the total number of culturable bacteria remaining after each cell lysis treatment (protocols 2-5) was examined for soil no. The results show that the number of cultivable bacteria does not decrease with the intensity of the cell lysis treatment (in all cases and even when no treatment is applied, such as according to Protocol 1, About 2 × 10 ⁶ cfu / g soil).

These low cfu values were probably due to the use of dry soil,
And probably only the most resistant bacteria grew on the plate. The number of actinomycetes that form colonies is generally higher than for total cfu (total bacteria). This is because the spore germination step included in the actinomycete detection protocol was lacking in the control of all the bacteria.

[0497]   2.5 Recovery of added lambda phage DNA   The purpose of these experiments was that continuous cell lysis treatment affected the recovery of naked DNA.
And how these continuous cell lysis processes contributed to its degradation
It was to evaluate whether or not.

The DNA is a fraction of extracellular DNA released from already dead organisms that can remain in the soil for months (Ward et al., 1990), or is immediately lysed during the first step of the treatment. Could be DNA released from the living organism. To simulate this situation,
Lambda phage DNA digested with HindIII was added to soil at various concentrations before and after grinding. In addition to crushing, other cell lysis treatment (sonication (C
up Horn device, see protocol 4b) and heat shock (see Materials and Methods section)) was tested.

After extraction, aliquot fractions theoretically required to contain 25-150 ng of lambda phage DNA were analyzed by gel electrophoresis. Regardless of soil type and quantity, it was possible to observe DNA fragments specific for the lambda phage when the DNA was inoculated into the soil sample before grinding.

When the DNA was added after milling and extracted without an additional cell lysis treatment step, the specific lambda phage DNA profile was detected in extracts of 4 out of 5 soils tested.

In all of these cases, a direct cause-effect relationship was obtained between the amount of DNA added and the intensity of the signal on the agarose gel. However, the signal intensity was lower than the signal intensity expected in comparison with the case of the molecular weight standard.

Furthermore, in some cases the 23 kb band was absent. This indicates that long fragments were preferentially adsorbed on soil particles or were more sensitive to degradation than short fragments.

No band was detected in the sample of tropical soil number 5 (Table 1), which is characterized by a very high clay content.

For more precise quantification, the amount of DNA recovered was measured on a phosphorescence imaging device (phosphoimager) after dot blot hybridization. This technique detected the DNA in all samples, including those inoculated prior to milling (except soil number 5 where no DNA was detected at all).

In all other soils, the amount of DNA extracted increases with increasing size of the inoculum (FIGS. 4a-d).

However, the amount of lambda phage DNA recovered was low. If crushing was the only cell lysis treatment applied, the recovery was 0.6-5.9% of the added DNA, if the DNA was added prior to crushing. When added after milling, it was 3.6-24% of the added DNA. The highest level of recovery was obtained from soil number 2.

On gel electrophoresis of aliquots of samples treated by heat shock and sonication, no DNA bands could be observed in any of the samples, including the test in which the DNA was added after grinding. The dot blot hybridization experiments confirmed these results.

Hybridization signals obtained from soil suspensions treated with heat shock and sonication were low at best.

The sample showing the highest amount of DNA (15 μg DNA / g soil dry weight) was the only one in which the signal obtained was substantially different from the background level.

No difference (or only small difference was observed) between the heat shock treated and heat shock and sonication treated samples. This indicates that heat shock has a deleterious effect on the DNA. The best recovery is soil number 2 with the highest organic matter content (Table 1
), While no DNA was recovered from clay soil number 5.

Additional experiments were performed with unground samples of soil # 4 and # 5 inoculated with 20 and 50 μg of lambda phage DNA per gram of soil.

The samples were extracted after or immediately after incubation for 1 hour at 28 ° C., then the DNA extracts were purified and analyzed (performed by gel electrophoresis).

The 1 hour incubation of soil # 4 after the inoculation differs qualitatively or quantitatively from that obtained without incubation or as already observed when the DNA is added after grinding. Did not give a profile.

These results indicate that enzymatic degradation by soil nucleases does not appear to be involved in low level DNA recovery. Furthermore, the absence of a milling step did not allow for an increased recovery of DNA from soil # 5, which means that changes to the structure of the soil by the milling significantly resulted in the adsorption of the nucleic acid on the colloid. It shows that it does not increase.

[0515]   2.6 Saturation of adsorption site by RNA   Most of the profiles obtained on the agarose gel were RNA treated.
It is not significantly different from the previous profile which was not missed.

For example, regardless of the lambda phage DNA and RNA concentrations used,
No band was detected from clay-rich soil No. 5.

Furthermore, in the sandy soil mixed with RNA (Soil No. 4), when the RNA was added before crushing, a specific band of HindII-digested lambda phage DNA could not be detected. It was as it was.

The intensity of bands obtained from samples inoculated with DNA after milling increases as the RNA concentration increases, indicating that the treatment may have a positive effect.

However, the results after analysis by hybridization and phosphorescence imaging did not prove their electrophoretic results. For example, the positive effect of the RNA treatment on the recovery of DNA from the clayey soil was not apparent when the DNA was added after grinding.

On the other hand, in clay-rich soil (No. 5), a positive effect of the RNA was found when the DNA was added after grinding.

The hybridization signal for the control sample does not differ from the background noise level, but from the sample treated with RNA, a significant amount of D
The signal increased as NA was released and the amount of added DNA increased and the RNA concentration increased.

However, even at the highest RNA concentration (100 mg / g soil dry weight), the recovery level never exceeded 3%.

[0523]   2.7 Purification of crude DNA extract   Of the four protocols tested, the undiluted DNA extract (50 μl PC
The best amplification of 1 μl of extract in R mixture) was on gel electrophoresis of the PCR product.
As shown, elution through the Mcrospin S400 column and its
Observed after subsequent elution through an Elutip d column.

DNA purified by the two-phase aqueous system (Protocol C) gave a smaller amount of PCR product after amplification starting from undiluted DNA extract.

No amplification product could be obtained from the undiluted extract after amplification after using protocols A or B. Therefore, protocol B (for all experiments involving the PCR amplification and / or the dot blot hybridization)
See Materials and Methods section).

[0526]   2.8 Quantification by PCR and hybridization   The first step is to determine the number of target DNA molecules initially present in the reaction tube by PCR production.
It was to determine whether the quantities of things were proportional. Streptosporangium
DN from Flasil (Streptosporangium fragile)
A was used as the target (see Materials and Methods section).

The primers used were primers FGPS122 and FGPS350 (Table 2). Gel electrophoresis of the PCR products showed that the band intensity increased with increasing concentration of the target. The PCR product was hybridized to the oligonucleotide probe FGPS643 (Table 2) and the signal was quantified by phosphorescence imaging (phosphoimaging).

A good correlation (r ² = 0.98) was found between log [number of targets] and log [strength of hybridization signal].

Studies were then conducted to determine if the efficacy of the PCR amplification was compromised by humic acid and non-target DNA. When analyzed by gel electrophoresis, the increased band intensities of the PCR product, corresponding to varying amounts of target DNA, showed that DNase containing humic acid at concentrations ranging up to 8 ng in 50 μl of the PCR mixture. It was maintained when the amplification was carried out with the DNA solution to which the treated soil extract was added.

At 20 ng humic acid in the PCR mixture, the band corresponding to the low level of target DNA disappeared and at 80 ng humic acid concentration and higher concentrations, no band was visible.

[0531] S. fragile DNA before amplification
Streptomyces hygroscopics (Streptomyces hy
Groscopicus) DNA and PCR in the range of 100 pg to 1 μg
When simulating the non-target DNA released from the soil microbiota in addition to 50 μl of the mixture, various amounts of target DNA from S. fragile yielded the expected amount of PCR product. Made it possible to supply.

[0532]   2.9 Quantification of indigenous soil actinomycetes after various cell lysis treatments   Purification Protocol D was applied, followed by PCR amplification as described above,
Strep in soil number 3 after extraction according to Rotocall 1, 2, 3, 5a and 5b
Actinomycetes belonging to the genus Streptosporangium
It was quantified (Fig. 5).

After crushing (Protocol 2), the amount of target DNA derived from this actinomycete was determined to be 2.5 ± by hybridization (dot blot) and radioimaging.
It was estimated to be 1.3 ng / g dry soil weight.

With regard to Streptomyces, the DNA content is 1
Assuming 0 fg / cell (Gladek et al., 1984), this value is approximately 2
． It corresponds to 5 × 10 ⁵ genomes. Similar values were obtained after the other cell lysis treatments (2.6 ± 1 for protocol 3 and 4b, respectively).
． 1 and 1.8 ± 1.3 ng DNA / g dry soil).

[0535]   2.10 Effectiveness of DNA recovery from soil pre-inoculated with bacteria   Three soils (Nos. 2, 3 and 5) were mixed with various concentrations of Streptomyces
Spores or hyphae of Streptomyces lividans
Inoculated (see Materials and Methods section). Amount of mycelium added to the soil (
Figure 6b) corresponds to the number of spores inoculated into the germination medium. About 50 of these spores
% Germinated. The exact number of cells in the hypha of the germinated spore was not measured. Shi
Therefore, the amount of spores and mycelium inoculated into the soil can be compared directly.
Can not.

For each soil sample, the specific target DNA released was counted using a combination of extraction protocol 6, purification method D and PCR amplification with dot blot hybridization and phosphorescence imaging (phosphoimaging). When the number of added spores exceeds 10 ⁵ for soil number 3 and number 5 and 10 ⁷ for soil number 2, the extracted DNA can be clearly distinguished from background noise (Fig. 6a).

When the mycelium was added, the extracted DNA was above the amount corresponding to 10 ³ spores / g soil for soil numbers 2 and 3, and 10 ⁷ spores / soil for soil number 5. g can be detected above the amount corresponding to soil (Fig. b).

Above the detection level, the hybridization signal increases with increasing amount of inoculated cells.

For the spore inoculum, a 100-fold increase in the number of inoculated cells was
This leads to a nearly 100-fold increase in NA yield. This increase is clearly lower than when the mycelium was inoculated, especially in soil Nos. 2 and 3 (Fig. 6).

In contrast, in the results obtained when lambda phage DNA was used as inoculum, when the bacterial cells were used as inoculum, the DNA was also found in the clay-rich soil ( No. 5). However, also for the latter inoculum, treatment with RNA increased the recovery of Streptomyces DNA from this soil for both spores and mycelia (
(Figure 6).

Inoculation of the soil with Bacillus anthracis cells gave recovery levels similar to those obtained for Streptomyces.

In addition, the level of DNA recovery from soil no.
Increased after treatment with NA.

[0543]   Example 2: Low molecular weight DNA (<10 kb using soil contaminated with lindane ) Library construction, and cloning and expression of the linA gene
  This example describes the construction of an E. coli DNA library.
. It involves cloning and cloning small genes from non-culturable microflora.
And expression.

Lindane is an organochlorine pesticide that is resistant to degradation and remains in the environment. Under aerobic conditions, dehydrochlorinase encoded by the linA gene catalyzes biodegradation and converts lindane to 1,2,4-trichlorobenzene. The linA gene has two strains isolated from soil, namely Sphingomonas paus mobilis (Spinogomonas pauc) isolated in Japan.
imobilis) (Seeno and Wada 1989; Imai et al., 19).
91; Nagata et al., 1993) and Rhodanobacter lindani nicla isolated in France.
Sticus) (Thomas et al., 1996, Nalin et al., 1999).

However, the degradation potential of lindane, as shown by free chloride ion assay and PCR amplification of the linA gene from soil contacted with lindane, appears to be more extensive in the environment (Biesiekierska-Galgue).
n, 1997).

[0546]   1. Direct extraction of soil DNA   Restch Centrifuge Grind with 6 Tungsten Beads on Dry Soil
Grind in a dough for 10 minutes. 10 grams of ground soil in 50 ml of TEN pH 9
P buffer (50 mM Tris, 20 mM EDTA, 100 mM NaC
l, 1% w / v polyvinylpolypyrrolidone) for 10 minutes
Hox homogenize.

After centrifugation at 4000 xg and 4 ° C for 5 minutes, the supernatant was washed with sodium acetate (3M
, PH 5.2) and with isopropanol and then taken up in sterile TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0). The extracted DNA was then loaded onto an S400 molecular sieve column (Pharmacia) and an Elutip d ion exchange column (Schleicher and
Purify on Schuell) according to the manufacturer's instructions and then store in TE.

[0548]   2. Extracted from the soil in the vector pBluescript SK- DNA library construction   Vector pBluescript SK- and DNA extracted from the soil
Of the enzyme HindIII and 10 units per μg of DNA, respectively.
And BamHI (Roche) (2 hours incubation at 37 ° C.
). The DNA is then added to T4 D at a rate of 1 enzyme unit per 300 ng of DNA.
By the action of NA ligase (Roche), ligate at 15 ° C. overnight (about
200 ng DNA insert and 100 ng digested vector). Ele
Electrocompetent Escherichia coli (Esche
richia coli) cells ElctroMAX DH10B ™ (Gi
bco BRL) was electroporated (25 μl) with the ligation mixture (2 μl).
F, 200 and 500 Ω, 2.5 kV) (Biorad Gene Pulses)
er).

After 1 hour incubation in LB medium, the transformed cells were diluted to obtain approximately 100 colonies / dish, and then ampicillin (100 m
g / l), γ-HCH (500 mg / l), X-gal (5-bromo-4-chloro-3-indolyl-α-D-galactoside, 60 mg / l) and IPTG (
Plate on LB medium (10 g / l tryptone, 5 g / l yeast extract, 5 g / l NaCl) supplemented with isopropylthio-β-D-galactoside, 40 mg / l) and incubate overnight at 37 ° C. Since γ-hexachlorocyclohexane (Merck-Schuchardt) is insoluble in water, D
A 50 g / l solution is prepared in MSO (dimethyl sulfoxide) (Sigma).

[0550]   In this way, a library of 10,000 clones was obtained.

[0551]   3. Cloning and expression of the linA gene   The library was screened for lindane-degrading halos around the colony.
Visualization was performed (lindane precipitated in the medium). Screened 10
Of the 1,000 clones, 35 showed lindene-degrading activity, thus
It was The presence of the linA gene within these clones was confirmed by Thomas et al. (1996).
) Was confirmed by PCR using the specific primers described in 1). On the insert
And the digestion performed on the amplification product
Reference control R. lindaniclas
Ticus) showed the same profile. ku that retains the linA gene
The loan also had an insert of the same size (about 4 kb).

As described above, it was shown that the soil DNA can be cloned and expressed in E. coli, which is a heterologous host, and that a gene derived from a microflora that is difficult to culture can be expressed. . Therefore, libraries prepared by partial digestion of DNA extracted from soil with a restriction enzyme such as Sau3AI can also be expected.

[0553]   Example 3   Method for producing a population of nucleic acids from a soil sample, including the step of direct DNA extraction   1.Materials and methods   1.1 Extraction of bacterial fraction in soil   Grind 5g of soil in Waring Blender for 3 x 1 minute (each grind
Between 50 ml of sterile 0.8% NaCl
Disperse into The bacterial cells were then passed through a Nycodenz density cushion (Nu
(comed Pharma AS, Oslo, Norway) by centrifugation.
Separated from the soil particles. In a centrifuge tube, 1.3 g. ml^-1The density of
Having 11.6 ml of Nycodenz solution (8 suspended in 10 ml of sterile water)
g Nycodenz) is placed under the already obtained 25 ml soil suspension
. Rotor with swing-out bucket (TST 28.38)
Centrifuge for 40 minutes at 10,000 xg in a rotor, Kontron) at 4 ° C
After that, the cell ring located in the interphase between the aqueous phase and the Nycodenz phase is removed.
Wash with 25 ml of sterile water and centrifuge at 10,000 xg for 20 minutes.
To do. The cell pellet was then treated with 10 mM Tris; 100 mMn EDTA (
pH 8.0) Take in solution.

Prior to dispersing the soil in a Waring Blender, a step of enriching the soil in a solution of yeast extract may be included, in particular to allow germination of the soil bacterial spores. For example, 5 g of soil is incubated for 30 minutes at 40 ° C. in 50 ml of a sterile solution of 0.8% NaCL-6% yeast extract. The yeast extract is removed by centrifugation at 5000 rpm for 10 minutes to avoid the formation of foam during the milling.

[0555]   1.2 Cytolysis of soil bacterial cells   Cell lysis of the cells in liquid medium and purification on a cesium chloride gradient   The cells were treated with 5 mg. ml^-1Lysozyme and 0.5 mg. ml^-1Acromo
10 mM Tris, 100 mM EDTA (pH 8) containing peptidase
． 0) Lyse cells in solution for 1 hour at 37 ° C. Then Lauryl Sarkosi
(1% final) and proteinase K (2 mg.ml^-1) At 37 ° C
Incubate for 30 minutes. The DNA solution was then added to Kontron 65.
Cesium chloride by centrifugation on a 13 rotor at 35,000 rpm for 36 hours.
Purify on a column density gradient. The cesium chloride gradient used has a refractive index of 1.3860.
Gradient with 1 g / ml of CsCl with (Sambrook et al., 1989).
).

[0556]   ・Cytolysis of the cells after addition in agarose block   The cells were treated with a low melting point of 1.5% (weight / volume) Seaplaque (Agaro
se Seaplace FMC Products. TEBU, Le Pe
equal volume agaro containing rray en Yvelines, France)
Pour into a 100 μl block. The block is then lysed
Solution: 250 mM EDTA, 10.3% sucrose, 5 mg. ml^-1  Re
Zozyme and 0.5 mg. ml^-1  3:00 in achromopeptidase at 37 ° C
Incubate for Then, the block was treated with 10 mM Tris-500 mM.
Wash in EDTA solution, lmg. ml^-1Proteinase K and 1% lauri
Incubate overnight at 37 ° C in 500 mM EDTA containing lusarcosyl.
To After several washes in Tris-EDTA, the block was washed with 500 mM.
Store in EDTA.

The amount of DNA thus extracted is confirmed by pulse field electrophoresis.

The amount of DNA extracted was evaluated on an electrophoretic gel against a calibration range of calf thymus DNA.

[0559]   1.3 Molecular characterization of DNA extracted from soil   The DNA extracted from the soil was characterized by PCR hybridization.
Kick The method comprises, in the first step, universally conserving the 16S rRNA gene.
The DNA is amplified using primers located on the isolated region, and then the amplified D
Hybridize NA to various oligonucleotide probes of known specificity (Table 4).
To the outside of the genomic DNA,
Includes quantifying the strength of the gnoll.

DNA extracted from the soil and genomic DNA extracted from pure culture were
Amplify with primers FGPS 612-669 (Table 1) under standard PCR amplification conditions. Then, the amplification product was denatured with an equal volume of 1N NaOH, and a Nylon membrane (GeneScreen Plus, Life Science Product) was added.
ts) and are hybridized with an oligonucleotide probe end-labeled with g ³² P by the action of T4 polynucleotide kinase. 6
After prehybridization of the membrane in 20 ml of solution containing 20 ml of SSC 20 ×, 1 ml of Denhardt's solution, 1 ml of 10% SDS and 5 mg of heterologous salmon sperm DNA, the hybridization is determined by the probe. Overnight at temperature. The membrane was washed twice in SSC 2x at room temperature for 5 minutes, then once in SSC 2x 0.1% SDS and once more SSC 1
X, wash in 0.1% SDS for 30 minutes at the hybridization temperature. The hybridization signal was analyzed by Molecular Anal.
yst software (Biorad, Ivy sur Seine, Franc
ce)), and the amount of DNA is evaluated by the interpolation method of the calibration curve obtained from the genomic DNA.

[0561]   2. Results and Discussion   2.1 Extraction and cell lysis of the bacterial fraction of soil   Separation of microbial cells from soil particles prior to extraction of DNA is carried out by
Is an alternative that has a number of advantages over the direct extraction method of. Especially,
Extraction of the product fraction was carried out by using the external DNA existing in the soil in a free manner or by the eukaryote-derived D
Prevents the DNA extract from being contaminated with NA. In particular, the microbial fraction of the soil
DNA extracted from Escherichia coli has a longer size than DNA extracted by direct cell lysis.
Fragments and better integrity (Jacobson and Rasmu
ssen (1992)). Furthermore, the separation of the soil particles is dependent on humus and phenol
Compounds contaminate the DNA extract and these compounds then
It makes it possible to avoid significantly damaging the.

One of the determinant steps in the extraction of cells from the soil is the dispersion of the soil sample to dissociate the cells that attach to the surface or inside the aggregates of soil particles. Three consecutive grinding cycles of 1 minute each allow to obtain better cell extraction efficiency and higher amount of recovered DNA than a single 1 minute 30 second grinding cycle.

Table 5 shows the total viable microbiota (count by microscopic examination after staining with acridine orange), total culturable microbiota after centrifugation on a Nycodenz gradient.
Solid 10% Trypticase-Soja medium) and a cultivatable actinomycete flora on HV agar (6% yeast extract-0.05% SDS solution to cause germination of the spores) Extraction efficiency on medium) (after incubation at 40 ° C.). In addition, the extracted DNA is included in the agarose block after cell lysis of the cells in liquid medium (without purification on a cesium chloride gradient) or (after digestion of the agarose with β-agarase). The cells were lysed and quantified.

The results show that over 14% of the total terrestrial microbiota is recovered by this method (ie 2 × 10 ⁸ cells / g soil) and that the total culturable microbiota is 2% of the total microbial population. It is equivalent to nothing but.

[0565] Furthermore, the amount of DNA extracted from the cells was 330 ng / g of dry soil.
Is. Estimating the DNA content per soil microbial cell to be 1.6 to 2.4 fg, and considering the amount of extracted cells (2 × 10 ⁸ cells per 1 g of soil), substantially all of the cells are cells. It can be assumed that it is lysed, and that this cell lysis does not bring much bias to this approach.

The pulsed field electrophoresis showed that DNA from soil extracted after Nycodenz and CsCl gradients could be up to 150 kb in size, and the agarose gel block cell lysis allowed extraction of fragments over 600 kb. It shows that it did.

These results demonstrate the advantage of this culture-independent approach for the construction of environmental DNA libraries as an alternative to the method of direct DNA extraction.

[0568]   2.2 Molecular characterization of DNA extracted from soil   The purpose of the molecular characterization of DNA extracted from soil is the presence in the DNA extract.
To obtain a profile representing the proportions of the various bacterial taxa. Also it
Is a direct extraction method without direct visualization of the microbial diversity present in the soil
The extraction bias induced by pre-separation of the cellular response of the soil compared to
Including that. In particular, their morphological structure (cell diameter, fibrous or sporulated form
Information on the extraction of cells on the Nycodenz gradient correlated with
Not collected.

Suitable methods so far are based on the following. -Quantitative hybridization using oligonucleotide probes specific for different bacterial groups, applied directly to DNA extracted from the environment. Unfortunately, this approach is not very sensitive and does not allow the detection of taxa or genera present in low abundance (Amann (1995)). -MPN-PCR (Most Probeable Number) (Syke
s et al. (1992)) or competitive quantitative PCR (Diviacco et al. (1993).
)) Quantitative PCR. The drawbacks of each of these approaches are (i)
The technique is not suitable for large numbers of samples or primer pairs due to the large number of dilutions and iterative efforts, and (ii) construction of competitors that are specific to the target DNA and does not induce bias in the competition. The point is.

The method introduced by the present invention is to universally amplify a 700 bp fragment within the 16S rDNA sequence, with oligonucleotide probes of different specificity (for kingdom, order, subclass or genus). Hybridizing this amplification product and comparing the hybridization intensity of the sample to an external calibration range. Pre-hybridization amplification allows quantification of relatively few microbial genera or species. Furthermore, amplification with universal primers allows the use of a wide range of oligonucleotide probes during the hybridization. It allows the comparison of different modes of cell lysis (direct or indirect extraction) for well-defined taxa.

[0571]   The results are compared in Table 6.

They show a similar profile between their two extraction methods (direct and indirect). Therefore, pre-extraction of aboveground microflora fractions does not appear to introduce a true bias between the taxa tested. The only significant difference between those two extraction approaches would be the greater abundance of rDNA sequences belonging to γ-proteobacteria in extracts by the indirect extraction method.

Furthermore, a significant effect of incubation of the soil sample in a solution of yeast extract is observed on the spore-forming soil population (gram ⁺ , low proportion of GC and actinomycetes). This step causes the spores to germinate, firstly allowing a better recovery of cells of this type, and secondly, allowing a greater cytolytic efficacy in the germinated cells. .

This approach allows semi-quantitative analysis targeted to major taxa defined using cultivated microorganisms normally found in soil. Only molecular means make it possible to assess the size of different taxa. This is because the culturing method is too limited and depends on the specificity of the medium used.

The results show that a large proportion of the microbial population is not represented in the described phylogenetic groups, which means that it has not been previously cultured or culturable. It shows the existence of a new group consisting of no microorganisms.

Therefore, in order to obtain a more rigorous picture of the composition of DNA extracts, given sequences starting from DNA extracted from soil can be used to define new probes (uncultured microorganisms). A new strain consisting of Ludwig et al. (19
97)).

[0577]   Example 4: Construction of cosmid POS700I   Features of POS700I   Replicates in E. coli.   Incorporated within Streptomyces.   E. coli AmpR, HygroR and Streptomyces (
Streptomyces) HygroR.

The properties of the cosmid allow the insertion of large DNA fragments of 30-40 kb. It includes the following: 1. Streptomyces lividance
ans) inducible promoter tipA. 2. An integration system specific to the element pSAM2. 3. Hygromycin resistance gene. 4. Cosmid pWED1 derived from pWED15.

[0579]   1) TipA remains of S. lividans Gene inducible promoter   The tipA gene is transcribed by the antibiotic thiostrepton or nosiheptide.
Encodes a 19 KD protein that is induced. tipA is well regulated
Induction (200 ×) in exponential phase and stationary phase (Murakami T, H
lt TG, Thompson CJ. J. Bacteriol 1989
171: 1459-66).

[0580]   2)Hygromycin resistance gene   ・ Hygromycin: Streptomyces hygroscopicus (S. hyg
antibiotics produced by Roscopicus.   -The resistance gene encodes phosphotransferase (hph).   -The genes used are the hyg gene's own promoter and IPTG.
Constructed by Blondelet et al. Under the control of the inducible plac promoter
Cassette (Blondelet-Rouault et al .; Gene 1997; 1).
90: 315-7).

[0581]   3)Site-specific integration system   Element pSAM2 is integrated into the chromosome by a site-specific integration mechanism. Embedded
The two clones present on the plasmid (attP) and on the chromosome (attB)
It occurs between identical 58 bp sequences.

The int gene, located near the attP site, is involved in the site-specific integration of pSAM2, the product of which has similarities to the integrase of the enterobacterial temperate bacteriophage. It has been shown that a pSAM2 fragment containing only the attP attachment site and the int gene can be integrated in the same manner as all elements (1
8/05/1988 French Patent No. 88 06638 and Raynal
A et al., Mol. Microbiol. 1998 28: 333-42).

[0583]   4) Construction of cosmid pOS700I   Process 1   Promoter TipA on a 700 base pair HindIII-BamHI fragment
Plasmid pPM927 (Smokvina et al., Gene 1990; 94:
53-9) and vector pU digested with HindIII / BamHI
It was cloned into C18 (Yannish-Perron et al., 1985).

Step 2 The HindIII-BamHI fragment was then transferred from pUC18 to pUC19 (
Yannish-Perron et al., 1985).

Step 3 1500 base pair Ba carrying the attP site of pSAM2 and the int gene
The mHI-BamHI insert was labeled with pOSint1 (Ray) represented in FIG.
nal A et al. Mol Microbiol 1998 28: 333-42).
And was cloned into the BamHI site of the previous vector (pUC19 / TipA) in an orientation that allowed the int gene to be placed under the control of the promoter TipA.

Step 4 The BamHI site located 5'to the int gene was deleted by partial digestion with BamHI followed by treatment with Klenow enzyme. In this way, T
The HindIII-BamHI fragment containing ipA-int-attP was pUC.
Isolated from 9 and introduced into pBR322 HindIII / BamHI.

Step 5 The hygromycin cassette isolated with the HindIII-HindIII fragment from pHP45Ωhyg (Blondelet-Rouault et al., 1997) was cloned into the HindIII site located upstream of the promoter TipA.

Step 6 The HindIII site located between the ΩHyg cassette and the promoter TipA was deleted by Klenow treatment after partial HindIII digestion.

Step 7 The plasmid obtained after the previous step was cloned into the EcoRV site of cosmid pWED1 after treatment with Klenow and all ΩHyg / TipA / int were cloned.
Allows the isolation of a single HindIII-BamHI fragment carrying the attP element. The cosmid pWED1 shown in FIG. 9 is the cosmid pWE shown in FIG.
15 (Wahl GM et al., Proc. Natl. Acad. Sci. USA 1
987 84: 2160-4) by the deletion of the HpaI-HpaI fragment retaining the neomycin gene and the SV40 origin.

[0590]   A map of the vector pOS7001 is shown in FIG.

[0591]   Example 5: Cosmids that are mating and integrating in Streptomyces , Construction of the vectors pOSV303, pOSV306 and pOSV307   5.1 Construction of vector pOSV303   Assuming packaging selects clones larger than 30 kb,
Only 10-15% of the clones contained no inserts,
And it is not really necessary to have a system for selecting recombinants,
This allows the construction of smaller vectors.

[0592]   Construction   Step 1: Vector pOSV001   The replicon RK2 (Gu) was inserted into the PstI-cleaved plasmid pUC19.
iney et al., 1983) 800 base pair PstI retaining the origin of introduction OriT.
-Cloning of the PstI fragment. This cloning step is carried out in E. coli (E.
Transferable from i) to Streptomyces by conjugation
It is possible to obtain a competent vector.

[0593]   A map of the vector pOSV001 is shown in FIG.

Step 2: Vector pOSV002 Streptomyces (so that the hygromycin resistance gene was introduced last)
Streptomyces) selectable hygromycin marker (
Ωhyg cassette) so that a complete introduction of BAC with the soil DNA insert can be guaranteed.

Cloning of the hygromycin cassette isolated from pHP45Ωhyg as a HindIII-HindIII fragment carrying the hygromycin resistance gene. This fragment is cloned into the PstI site (position 201) of vector pOSV001. This PstI site was selected considering the direction of the introduction so that the Hygro marker was introduced last during the conjugation. PstI and H
The indIII ends become compatible after treatment with the Klenow fragment of DNA polymerase to generate "blunt ends". The orientation of the Ωhyg fragment is determined at the end of construction.

[0596]   A map of the vector pOSV002 is shown in FIG.

Step 3: Vector pOSV010 The XbaI-HindIII fragment isolated from the plasmid pOSV002 and containing the hygromycin resistance marker and the origin of introduction was digested with XbaI and Hind.
Clone into the plasmid pOSint1 digested with III. The orientation of the site is such that the hygromycin marker is always introduced last.

The plasmid pOSint1 shown in FIG. 8 is obtained by the method of Raynal et al. (Ray).
nal A et al., Mol. Microbiol. 1998 28: 333-42).
It is described in.

This construct was used in E. coli and Streptomyces (Str.
It enables the expression of integrase in ptomies).

Step 4: Insertion of "cos" Site The principle is to insert a "cos" site into plasmid pOSV010 to allow packaging into plasmid pOSV010 shown in FIG.

[0601]   The production of the "cos" fragment is shown in Figure 13.

This fragment is obtained by PCR. Starting from a fragment retaining the cohesive end (cos) of λ (bacteriophage lambda or cosmid pHC79), PC using an oligonucleotide corresponding to the sequence −50 / + 130 to the cos site
Perform R amplification. These oligonucleotides also contain NsiI cloning site, PstI (compatible), XhoI site, SalI (compatible) and EcoRV (
Sites for obtaining "blunt ends").

Addition of rare SwaI and PacI sites allows isolation and / or mapping of cloned inserts.

The PCR fragment had a PstI site at the 5 ′ end and a HincI site at the 3 ′ end.
It is delimited by the I site and can be cloned into the vector pOSV010 (FIG. 12) which has been predigested with the enzymes NsiI and EcoRV so that a deletion of the laclq repressor occurs.

A map of the vector pOSV303 is shown in FIG. The vector pOSV303 is
It contains cloning sites such as the NsiI site, PstI (compatibility), XhoI site, SalI (compatibility) or EcoRV site (the site for obtaining “blunt ends”).

[0606]   5.2 Construction of vector pOSV306   Step 1: Construction of vector pOSV308   The vector pOSV308 was constructed according to the method described in FIG. Array array number
No. 107 and the pair of primers of SEQ ID NO.
And the cosmid vector pHc79 described in Collins (1980), c
A 643 bp fragment containing the os region was amplified.

This amplified nucleotide fragment was labeled with Promega as shown in FIG.
Was directly cloned into the pGEMT-easy vector sold by the company to obtain the vector pOSV308.

[0608]   Step 2: Construction of vector pOSV306   As described in step 3 of the construction of the vector pOSV303 described in section 5.1 of this example.
The vector pOSV010 was constructed as follows.

Vector pOSV10 was digested with the enzymes EcoRV and NsiI to obtain 7874b.
The p fragment was excised and then purified (as described in Figure 28).

Next, the vector pOSV308 obtained in the above step 1) was digested with the enzymes EcoRV and PstI to excise a 617 bp fragment, which was then purified.

The 617 bp cos fragment obtained from vector pOSV308 was then ligated into vector pOSV10 to give vector pOSV306 (FIG. 2).
8).

[0612]   5.3 Construction of vector pOSV307   Cosmid pOSV307 is a product of Streptomyces.
), For example to the S17-1 strain of Streptomyces
It still contains the Laclq gene to improve the stability of the cosmid.

To construct the vector pOSV307, the vector pOSV010 was transformed with the enzyme P.
Digestion with vuII gave a 8761 bp fragment which was purified and then dephosphorylated.

Next, the vector pOSV30 obtained as described in step 1) of section 5.2 above.
8 was digested with the enzyme EcoRI to give a 663 bp fragment which was then purified and treated with Klenow enzyme.

The nucleotide fragment thus treated was ligated to the vector pOSV01 after ligation.
Incorporation into 0 gave vector pOSV307 (as shown in Figure 29).

[0616]   Example 6: Escherichia coli-Streptomyces replicative shuttle cosmid pOS700 Construction of R   Isolation of a fragment of plasmid pEI16 (Volff et al., 1996) shown in FIG.
And treated with Klenow. These fragments are replicative and derived from plasmid SCP2.
And contains the sequences required for stability.

These two fragments are inserted separately into the EcoRV site of cosmid pWED1 to give two different clones.

The hygromycin cassette isolated from pHP45Ωhyg as a HindIII-HindIII fragment was cloned into S in the form of PstI-EcoRI or XbaI.
It was cloned into the HindIII site of the pWED1 cosmid containing the cP2 insert. It includes E. coli and Streptomyces (S.
confers hygromycin resistance, which can be selected for in both treptomies.

Transformation of S. lividans and Measurement of Transformation Efficiency Cosmids containing the XbaI insert were found to be less stable than those containing the PstI EcoRI fragment. Therefore, the latter cosmid (
designated as pOS700R).

[0620]   A map of the vector pOS700R is shown in FIG.

[0621]   Example 7: Transformation efficiency of integrative (pOS700I) and replicative vectors   possibility   By incorporating the plasmid pTO1 carrying the thiothrepton resistance marker
And Streptomyces lividans thiothrepto
Resistance to

Streptomyces lividans (S. elegans) cultured in the presence of thiostrepton.
Preparation of protoplasts from lividans).

With the vector pOS700I, the transformation efficiency is about 3000 transformants per μg of DNA.

With the vector pOS700R, the transformation efficiency is about 30,000 transformants per μg of DNA.

[0625]   Example 8: BAC vector that is a mating type integrated in Streptomyces -Building   Characteristic: It is replicated in E. coli. E. coli with Streptomyces
Can be introduced by joining. Incorporated in Streptomyces. E. coli and Streptomyces
). Large DNA fragments can be inserted. 100 ~ not contaminated by small pieces
It should be pointed out that 300 kb size of soil DNA needs to be available
Is. The reason for this is that such small pieces are very easy to integrate.
. It may be screened to select for plasmids carrying the insert.
This screen removes self-closed and undigested vectors
Thereby allowing a higher ratio of the vector and the inserted DNA to be achieved,
It may give better cloning efficiency for the production of libraries.

[0626]   Construction:   Step 1: Vector pOSV001   The replicon RK2 (Gu) was inserted into the PstI-cleaved plasmid pUC19.
iney et al., 1983) 800 base pair PstI retaining the origin of introduction OriT.
-Cloning of the PstI fragment. This cloning step is carried out in E. coli (E.
Transferable from i) to Streptomyces by conjugation
It is possible to obtain a competent vector.

[0627]   A map of the vector pOSV001 is shown in FIG.

Step 2: Vector pOSV002 Streptomyces (so that the hygromycin resistance gene was introduced last.
Streptomyces) selectable hygromycin marker (
Ωhyg cassette) so that a complete introduction of BAC with the soil DNA insert can be guaranteed.

Cloning of the hygromycin cassette isolated from pHP45Ωhyg as a HindIII-HindIII fragment carrying the hygromycin resistance gene. This fragment is cloned into the PstI site (position 201) of vector pOSV001. This PstI site was selected considering the direction of the introduction so that the Hygro marker was introduced last during the conjugation. PstI and H
The indIII ends become compatible after treatment with the Klenow fragment of DNA polymerase to generate "blunt ends". The orientation of the Ωhyg fragment is determined at the end of construction.

[0630]   A map of the vector pOSV002 is shown in FIG.

Step 3: Vector pOSV010 The XbaI-HindIII fragment isolated from plasmid pOSV002 and containing the hygromycin resistance marker and the origin of introduction was digested with XbaI and Hind.
Clone into the plasmid pOSint1 digested with III. The orientation of the site is such that the hygromycin marker is always introduced last.

The plasmid pOSint1 shown in FIG. 8 is obtained by the method of Raynal et al. (Ray).
nal A et al., Mol. Microbiol. 1998 28: 333-42).
It is described in.

This construct was constructed in E. coli and Streptomyces.
It enables the expression of integrase in ptomies).

[0634]   Step 4: Vector pOSV014   Selecting a plasmid with foreign DNA inserted in the final construct
The addition of a "cassette" that makes it possible at the end.

This "cassette" carries the gene encoding the lambda phage Cl repressor and the tetracycline resistance gene. This gene retained the target sequence of the repressor within its non-coding 5'region. H located within the code region of Cl
Insertion of DNA within the indIII site results in the absence of repressor production and thus the development of tetracycline resistance.

It is described by Nilsson et al. (Nucleic Acids Res. 1).
983, 11: 801-30) and is held by the plasmid pUN99.

Sequences Int, attP, Hygro and ori isolated from pOSV010.
The PvuII-HindIII fragment containing T is cloned into the MscI site of pUN99.

[0638]   A map of the vector pOSV014 is shown in FIG.

[0639]   Step 5: Vector pOSV403 and integrative and mating BAC vectors -   This final step of cloning into pBAC11 (shown in Figure 20)
Properties of the final plasmid BAC (bacterial artificial chromosome), especially very large DNA
Gives the acceptance capacity of sart.

The vector pOSV01 carrying the previously described set of elements and functions.
The PstI-PstI fragment of 4 was digested with the NotI plasmid pBAC11.
Clone in (pBeloBAC11). The ends are made compatible by treatment with Klenow enzyme.

A map of the vector pOSV403 is shown in FIG. The scheme of Figure 21 shows the selected orientation.

Step 6 : Vector pOSV403 contains HindIII and NsiI sites. The NsiI site is very rare in Streptomyces and has the advantage of being compatible with PstI. on the other hand,
The PstI site is common in Streptomyces and can be used to perform partial digestion.

Recombinant clones harboring an insert cloned into the Cl repressor that inactivates this repressor become tetracycline resistant. The BAC
Assuming that is present at only 1 copy / cell, it is necessary to select recombinant clones with tetracycline doses lower than usual (eg 5 μg / ml) of 20 μg / ml. Under these conditions there is no background noise.

It is also possible to use the system developed and sold by InVitrogen, in which case the insertion of the DNA into the vector is toxic to E. coli when expressed. Inactivate the gyrase inhibitor. The fragment is the vector pZErO-2 (http: //www.invitro.
gen. com /).

[0645]   Example 9: Embedded cosmid (pOS700I) and replicative cosmid (pO) Construction of Streptomyces alboniger library in S700R)   1) Library construction   To evaluate the effectiveness of the cloning system, Streptomyces arbonii
Raw Puromycin from Garr (Streptomyces alboniger)
The synthetic route was integrated into the two shuttle cosmids pOS700I and pOS700R.
I've roasted. The puromycin biosynthetic pathway gene has a Bam of about 15 kb.
It is retained by the HI DNA fragment.

[0646] Streptomyces alboniger
iger) genomic DNA was isolated. 90% of this DNA has a molecular weight of 20-150 kb as measured by pulse field electrophoresis.

The two cosmids were digested with the enzyme BamHI (single cloning site).

The conditions for partial BamHI digestion of genomic DNA were determined (50 μg DNA and 12 units enzyme, 5 min digest). After confirming the size by agarose gel electrophoresis, the partially digested DNA was introduced into the vector. In the ligation, 15 μg of genomic DNA + 2 μg of the integrative vector or 5 μg of the replicative vector was used.

For the in vitro encapsulation of the DNA into the head of bacteriophage lambda,
Each ligation mixture was used. The envelope mixture (0.5 ml) was titrated (integrated vector pOS700I = 7.5 × 10 ⁵ cosmid / ml, replicative vector =
5 × 10 ⁴ cosmid / ml).

The cosmid was used to transfect E. coli, thereby creating a library of approximately 25,000 ampicillin resistant clones.
DNA from all of these clones was isolated and quantified.

To test the library, several clones were selected, the DNA was purified and digested with BamHI to confirm the presence and size of the insert.
The clones tested were 20-35 Kb of Streptomyces alboniger (S
． alboniger) insert.

[0652]   2) Identification of clones containing the puromycin biosynthetic pathway   Clones tending to contain the complete puromycin biosynthetic pathway were cloned into
A 1.1 kb pac gene that is a romycin resistance gene (Lacall et al., G
1989; 79, 375-80).
It was identified by zation.

[0653]   Library created in integration vector pOS700I   Of the 2000 clones analyzed, 9 clones hybridize to the probe.
Redistributed, they contain an insert of approximately 40 kb.

[0654]   Library prepared in replication type vector pOS700R   Of the 2000 clones analyzed, 12 clones were
Bridging, they contain an insert of approximately 40 kb.

Tercero et al. (J. Biol. Chem. 1996; 271, 1579-.
Using the data published by 90), clones containing the entire biosynthetic pathway were identified after hybridization with the appropriate probe. Some integrative and replicative cosmids contain a 12,360 base pair fragment after ClaI-EcoRV digestion, leading to the hypothesis of inserts containing the entire puromycin biosynthetic pathway.

[0656]   4) Confirmation of puromycin production by resistant clones (Rhone-Pou lenc)   a) Materials and methods   Strains and culture conditions:   Three resistant clones were selected to confirm the production of puromycin.
They are either inserts or duplicates in the integrative vector pOS700I (G20).
Streptomyces containing inserts in the molding vector (G21 and G22)
Corresponds to the S. lividans recombinant.

A reference strain was used to ensure that the medium used allowed this production. They are the S. alboniger wild-type strain ATCC 12461 which produces puromycin, and the plasmid pRCP-11 (Lacalle et al., 1992, the EMBO journal).
al, 11, 785-792) containing a complete puromycin cluster cloned into S. lividans.
) A recombinant strain (G23).

[0658]   The strain was inoculated into a medium having the following composition.

Organotechnie Bacteriological Peptone 5 g / L Final Medium Springer Yeast Extract 5 Liebig Meat Extract 5 Prolabo Glucose 15 Prolabo CaCO ₃ (1) 3 Prolabo NaCl 5 Difco Agar (2) 1. (1) 3 g of carbonate is mixed with 200 ml of distilled water and then sterilized separately. The addition is performed after sterilization. (2) The agar is pre-melted in 100 ml of distillation and then it is added to the other components of the medium. Adjust pH to 7.2 before sterilization. Sterilize at 121 ° C for 25 minutes.

50 μg / l hygromycin and 5 μg / l thiothrepton were added to the medium after sterilization to maintain selection pressure for cloning containing inserts with the marker gene present on the vector (the thiothrepton resistance gene). Is carried by the plasmid pRCP11).

50 ml of liquid medium dispensed in a 250 ml conical flask are inoculated with 2 ml of an aqueous suspension of spores and mycelia of each of the strains. 220 rp of the culture
Incubate at 28 ° C for 4 days with agitation at m. Then 50 ml of the production medium dispensed in a 250 ml conical flask are inoculated with 2 ml of these precultures. The production medium used was pristinamyc.
in) is an industrial medium (medium RPR201) optimized for production. The culture is incubated at 28 ° C. with 220 rpm agitation. After various incubation times, the conical flask of each culture is brought to pH 11 and then extracted twice with 1 volume of dichloromethane. The organic phase is concentrated to dryness under reduced pressure and then the extract is taken up in 10 μl of methanol. For the detection of puromycin, 100 μl of the methanol solution are analyzed by HPLC with a diode bar detector in a water-acetonitrile 0.05% TFA V / V gradient system on a C18 column.

[0662]   b) Results   Comparative HPLC analysis from cultures of various strains showed that in cultures of wild type strains 24
Demonstrate production of puromycin at incubations over time
. 48 hours in culture of clone G20 containing cosmid pOS700I
Production was clearly detected, though to a lesser extent, at intervals of time or more (FIG. 23)
. Contains the complete operon encoding the compound in plasmid pRCP11
The amount of puromycin was also detected in the clone G23. However
The culture of clones G21 and G22 containing cosmid pOS700R
However, no production was observed. The results are shown in FIG.

[0663]   c) Conclusion   The results obtained are based on the cloning system developed in cosmid pOS700I.
The complete biosynthetic pathway of Streptomyces lividans (S.
for expression under the control of its own regulatory sequences in a heterologous host such as
Can show effectiveness. In addition, these data also indicate that for puromycin
Demonstrate efficacy of screening of obtained library based on resistance of said clone
is doing. Because it is related to the resistance gene from a few clones
This leads to the identification of recombinants capable of expressing the biosynthetic pathway. So
The absence of puromycin production in other clones of M.
Although it contains a gene, any regulation, transduction or transfer necessary for the synthesis of the compound.
Explained by cloning only part of the operon lacking the sequence
Will.

[0664]   Example 10: Cloning of soil DNA into a vector   1) Preparation of soil DNA to be cloned   Various DNA fragments need to be purified depending on their use.

[0665]   Cosmid   The size of the molecule should be 30-40 kb. At this stage, from the soil
The extracted DNA is heterogeneous in size, up to 200 or 300 kb
Including the molecule. To homogenize the size, pass the solution through a 0.4 mm diameter needle.
The DNA is mechanically destroyed by passing it. In the size range of 30 kb
The fragments are unaffected by these repeated passages within the needle and are therefore particularly sized.
There is no need to perform separation based on. Because the packaging in the particles is
This is because those short inserts are automatically excluded.

[0666]   BAC   DNA preparation   Pulsed under conditions where 100-300 kb fragments are concentrated in a band of approximately 5 mm
The soil DNA is separated by field electrophoresis (CHEF type). this is,
In a gel containing 0.7% regular agarose or 1% low melting agarose
At a pulse time of 100 seconds and a temperature of 10 ° C for 20 hours.
Is obtained by

[0667]   DNA recovery   Two methods are used. Their selection is based on the size of the molecules to be separated, 1
It depends on whether it is up to 50 kb or higher.

[0668]   Up to 150 kb   Porosity of 0.7% agarose gel when no ethidium bromide is present
In particular, it allows the DNA to flow out by electroelution. This DNA is then
In order to avoid mechanical fragmentation of the molecule, a hydrophobic enlarged orifice pipetting device
It is handled using a table.

[0669]   100-300 kb   A band containing a fragment with a size of 100-300 kb is cut out. For the migration
A gel containing 1% low melting point agarose is used. This characteristic is that the DNA
Melting the gel at an acceptable temperature of 65 ° C. and then
Supply (Agarase sold by Boehringer)
Allows digestion at a temperature of 45 ° C according to the manufacturer's instructions.

[0670]   2) Built-in cosmid pOS700I and replication type cosmid pOS700R use   Construction with poly A poly T tail   principle   A cosmid vector cleaved at either cloning site was used as a single species (mon
modified at the 3'end by the addition of
. In addition, the DNA to be cloned is paired with a polynucleotide that can pair with the polynucleotide.
Modification at the 3'end by the addition of a seed polynucleotide.

Vector-fragment combinations to be cloned are made with these polynucleotides. The cos sequence of the vector allows for in vitro packaging of the DNA into lambda phage capsids.

[0672]   Vector preparation   The vector used is self-replicating in E. coli and is a streptoma.
It is a vector that is integrated in Streptomyces.

Selection is performed for ampicillin resistance in E. coli and for hygromycin resistance in Streptomyces. The cosmid is cleaved at one of two possible sites (BamHI or HindIII) and the 3'end is extended with a terminal transferase with poly A under conditions where the enzyme supplier anticipates the addition of 50-100 nucleotides. Let

[0674]   Preparation of DNA to be inserted   Under the conditions that bring the 3'end of the DNA into an extension comparable to that of the vector,
Extend with poly T with terminal transferase. Described by the manufacturer
Under the experimental conditions described, the poly A poly T tail is 30 to 70 bases long.

[0675]   Molecular assembly and in vitro encapsulation   To assemble the molecules, mix one vector molecule for each DNA molecule to be inserted.
To meet. The concentration of the DNA based on the mass is 500 μg. ml^-1Is.

The mixture is enveloped. The transfection efficiency depends on the strain used as recipient and the DNA to be inserted and is zero for the test DNA and the strain DH5α, which is comparable to the SURE and DH10B strains, but when extracted , The DNA yield is higher in the strain DH10B.

[0677]   Construction by dephosphorylation   Removing the overhanging 3'sequence and filling in the overhanging 5'sequence in the soil DNA
More blunt ends. This operation involves Klenow enzyme, T4 polymerase,
Performed with nucleotide triphosphates. The cosmid vector was digested with BamHI and
Digest with Klenow enzyme to blunt the ends and then dephosphorylate to self-close it.
Prevent the ring. After ligation, the mixture is enveloped and transfected (see previously described).
Do exactly what you want).

[0678]   3) Use of pBAC   principle   The conjugative and integrative plasmid pBAC contains HindIII and N
Contains the siI site as a cloning site. DNA sequences within these sites
Insertion of lambda phage CI represses the expression of the tetracycline resistance gene.
Inactivate the presser. Therefore, inactivation of the repressor will
This antibiotic (5 μg.ml^-1) Against. In these areas
Cloning is carried out by modifying the vector to prepare cloned DNA.
Be promoted.

[0679]   Vector preparation: HindIII example   Modifying the HindIII site to prevent the vector from self-closing.
To do. The first base (A) was reinserted to create a protruding 5'sequence that could not pair with the counterpart.
Let it form. The operation is performed with Klenow enzyme in the presence of dATP.

Successful manipulation is confirmed by self-ligation of the vector before and after treatment with the Klenow enzyme. With an equal amount of test DNA, 3000 clones are obtained before the treatment and 60 clones are obtained after the treatment.

[0681]   Preparation of DNA (100-300 kb size)   Add blunt ends to DNA   By removing the overhanging 3'sequence and filling in the overhanging 5'sequence, the DNA
Blunt ends. This operation involves Klenow enzyme, T4 polymerase,
Performed with nucleotide triphosphates.

[0682]   Preparation of the end: HindIII example   Addition of DNA to the vector recognizes the HindIII modified sequence of the vector
The oligonucleotide is used. They allow subsequent cloning
It contains a rare restriction site (SwaI; NotI). This technology is Elledg
e SJ, Mulligan JT, Ramer SW, Spotswood
  M, Davis RW. Proc. Natl Acad. Sci. USA 1
991 Mar 1; 88 (5): 1731-5. Two
Of complementary oligonucleotides are used. Oligo 1: 5'-GCTTATTTAAATATTAATGCGGCCGCCC
GGG-3 '(SEQ ID NO: 25). Oligo 2: 5'-CCCGGGCGGCCGCATTAATAATTTAAATA
-3 '(SEQ ID NO: 26).

After hybridization, they are phosphorylated at the 5'end with T4 polynucleotide kinase in the presence of ATP. This phosphorylation step can be omitted by using already phosphorylated oligonucleotides. The ligation of this double-stranded adapter with the DNA to be inserted into the vector is carried out in the presence of a very large excess of adapter (100 adapter molecules per inserted DNA molecule).
4 ligases at 14 ° C. for 15 hours. The excess adapter is removed by agarose gel electrophoresis and the molecule of interest is recovered from the gel by hydrolyzing it with agarase or by electroelution.

Vector-DNA Ligation The ligation is performed with 10 molecules of vector per insert molecule for 15 hours at 14 ° C.

[0685]   Transformation   The recipient strain is strain DH10B. The transformation is by electroporation
To do. In order to develop tetracycline resistance, the transformant was treated with antibiotics.
Incubate for 1 hour at 37 ° C. in medium containing no. 5 μg. ml^-1Te
By culturing on gelled LB medium supplemented with tracycline overnight, the
Select a loan.

[0686]   Example 11: Clone to clone attachment between E. coli and Streptomyces. Combined   E. coli strain S17.1 containing pPM803 and streptoma
Isses Lividans TK21
Join between   Introduction   Escherichia coli (E. coli) and Streptomyces
It is possible to perform conjugation between and (Mazodier et al., 1989). Wow
The drop technique (in which the recombinant vector is
10 μl of E. coli culture with 1 drop of Receptor Streptomyces
This by advancing the libidos (mixed with S. lividans)
Application of the method involves performing transformation of clones from clone to clone, while
At the end of the work, all of the libraries constructed in E. coli were
Be sure to be introduced in S. lividans
To do so. The library in E. coli is Streptomyces
Make sure it is perfectly represented in S. lividans
In order to do so, a large amount of transformation inevitably requires Streptomyces (Strep).
will result in the growth of transformed clones. Moreover, this method
Easy to automate.

[0687]   Preliminary test   E. coli strain S17.1 containing the vector pOSV303 and
Joining with S. lividans TK21
.

Under these conditions, 6 × 10 ⁶ E. coli cells were mixed with 2 × 10 ⁶ pre-germinated S. lividans spores in a final volume of 20 μl. To do.

[0689]   Development of the method   DNA extracted from certain actinomycetes must be modified and, as a result, must be restricted.
For example, it is known that it cannot be introduced into certain E. coli. these
E. coli strain DH10B, which receives E. coli DNA, contains only oriT.
Transfer the plasmid with Streptomyces
No, therefore the construction of such a plasmid is necessary. RP4 derivative
Should be introduced into it by integration into the chromosome, the derivative being
All the machines necessary to ensure the introduction of recombinant clones containing the origin oriT
Noh can be given in trance.

[0690]   Example 12: Cosmid in E. coli and Streptomyces lividans Library construction: cloning of soil DNA   The purpose is to identify bacteria (or other bacteria) for which culture methods under standard experimental conditions are unknown.
A preliminary step of culturing the microorganism in order to obtain the metabolic genes of
To construct a large size library of environmental DNA without doing anything.

Escherichia coli was prepared using the described method.
A DNA library in. This is an E. coli-S. Lividans shuttle cosmid pO.
It was carried out using S700I and DNA extracted and purified from the soil bacterial fraction. This last method makes it possible to obtain highly pure DNA with an average size of 40 kb. Also, in order to avoid partial digestion of the extracted DNA in the cloning, another method was adopted, which was based on the use of terminal transferase enzyme to add a polynucleotide tail to the 3'end of the DNA and to the vector. .

5 μg of DNA was treated with 60 mg of “Sai according to the protocol described in Example 3.
nt-Andre coast "soil extracted and terminal transferase (Ph
armacia) to extend the 3'end with a single polynucleotide (polyT) (Example 10).

The integrated cosmid pOS700I is manufactured according to protocol B1, Orsay. After a standard purification step in the presence of phenol / chloroform, one molecule of vector and one molecule of insert DNA are mixed to assemble the DNA and the vector. The mixture is then encapsulated within the head of a lambda bacteriophage (Amersham kit) useful for transfecting E. coli DH10B. The transfected cells are then seeded on LB agar in the presence of ampicillin (for selecting recombinants resistant to this antibiotic).

A library of approximately 5000 ampicillin resistant E. coli clones was obtained. Each clone is inoculated into microplate wells (96 wells) in LB or TB medium + ampicillin and stored at -80 ° C.

The sequence of the site of insertion of the soil fragment into the vector pOS700I created during the construction of the library was analyzed. To this end, cosmid 17 of the library
Were purified and the HindIII cloning site and Bam present in the vector
Primer sequence 5'CCGCGAATTC that hybridizes with the HI site
Sequencing with TCATGTTTGACCG 3 '.

The resulting sequence has a homopolymer tail length of 13-60 at the binding site.
It was possible to deduce that it was very diverse with poly-dA / dT. Except for the tail, the sequences of soil fragments thus produced have a G + C ratio of 53-70%. Although such ratios were unexpected, similar results have already been reported for crude preparations of soil DNA (Chatzinotas A et al., 1998).

A method of "pooling" 48 or 96 clones was used to analyze microbial and metabolic richness. Then these cloned "
PCR or hybridization experiments were performed using cosmid DNA extracted from the "pool".

[0698]   Example 13: Diversity of 16S ribosomal DNA in cloned DNA   a) Materials and methods   The cosmid of the library was extracted from the pool by alkaline lysis and
And cosmid DNA in supercoiled form after purification on a cesium chloride gradient.
Escherichia coli stain that can interfere with the study
Chromosomal DNA is removed.

After linearization of the cosmid by the action of S1 nuclease (50 units, 37 ° C. for 30 minutes), the 16S rDNA sequence contained in the clone pool was labeled with Mar.
universal primer 63f (5'-C defined by chesi et al. (1998).
AGGCCTAACACCATGCAAGTC-3 ') and 1387r (5'-
Amplification under standard amplification conditions using GGGCGGGWGTGTACAAGGC-3 '). Approximately 1.5 kilobases of the amplified product was converted to
Qiagen) and then E. coli (Escherichia c)
cloning directly into the vector pCRII (Invitrogen) in oli) according to the manufacturer's instructions. The insert is then replaced with the vector pCR
Amplify using primers M13 Forward and M13 Reverse specific for the II cloning site. Amplification product of the expected size (about 1.7 kb)
RFLP (using 0.1 units) of the enzymes CfoI, MspI and BstUI (
Restriction Fragment Length Polymorph
Hism) to select clones to be sequenced. The restriction profile obtained was converted to 2.5% Me containing 0.4 mg / ml ethidium bromide.
Separation on tapore agarose gel (FMC Products).

Next, the PCR product purified by the “Qiaquick gel extraction” kit was treated with N
The 16S rDNA sequence is directly determined using with the sequencing primers defined by Ormand (1995). Ribosomal Dat
base Project (RDP) database, version 7.0 (Ma
by comparing the prokaryotic 16S rDNA sequence matched in idak et al. (1999)) with the sequence with the SIMILARITY MATCH program, which allows obtaining similarity values for the database sequences. A developmental analysis is obtained.

[0701]   b) Results   To measure the phylogenetic diversity expressed in the library, the 1
47 sequences of 6S rRNA gene were isolated from a pool of 288 clones
, Almost completely sequenced. The results are shown in Table 7.

Analysis of the sequence by querying the database revealed that most (> 61% of the sequences were
) Has a percent similarity of 95% or less to the identified bacterial species (Table 7). Twenty-eight of the 47 sequences analyzed had uncultured bacteria as their closest relatives, and these sequences were DN extracted from the environment.
Obtained directly from A. Furthermore, the majority of these sequences have very low similarity (
88-95%), so 17 of the 28 sequences differ by more than 5% relative to their closest analogs.

Of the sequences that can be classified into a phylogenetic group, the majority of the sequences belong to the subclass Proteobacteria a (18 sequences with 89-99% similarity). The second group of sequences is represented by the Proteobacteria subclass g, which contains 9 sequences with 84-99% similarity. The groups of b-proteobacteria and d-proteobacteria are the Pharmicutes phylum with low G + C% and high G + C%, respectively, containing 1, 4, 3 and 5 sequences, respectively. One array (array a22
． 1 (19)) alone could not be classified into the defined major bacterial taxa. Its closest analog, Erothermobacter marianas (A
erothermobacter marianas (with 89% similarity) itself is a strain isolated from a marine environment that is not currently classified. Finally, the 6 sequences can be classified into the group Acidobacterium / Holophaga. This group consists of two cultivated bacteria, Acidobacteria capsulatum.
ulatum) and Holofaga foetida (Holophaga foeti)
This whole group has the unique feature that it is represented by only da) and that only 16S rRNA gene was detected by amplification and cloning using DNA extracted from environmental samples (mainly from soil). It is composed of bacteria (Ludwig et al., (1997)). The low similarity values between the various sequences that make up this group make it possible to predict great heterogeneity and diversity within this group.

[0704]   A series of results are shown in Table 7.

These results suggest that the sequences contained in the cosmid library are not only derived from phylogenetically diverse microorganisms, but especially from microorganisms that have not been isolated to date. Is shown.

The results of sequencing the amplified DNA have allowed the establishment of a phylogenetic tree of organisms whose soils in which the characterized sequence is novel are present.

The phylogenetic tree shown in FIG. 7 is based on MASE software (Fauner and Jura).
k, 1988) and modified by the Kimura 2-parameter method (1980) and the Neighbour Joining algorithm (Saitou and Nei, 1987). The phylogenetic analysis was performed on 16 cloned clones in the soil DNA library.
S rDNA sequence and BLAST 2.0 software (Atschul et al., 1
997) by Ribosomal Database Project (RD
P) database (version 7.0, SIMILARITY-MATCH program, Maidak et al., 1999) and comparison with the sequence of prokaryotic 16S rDNA matched in the GenBank base.

[0708]   Example 14: Genetic preselection of the library to assess metabolic enrichment   To characterize the resulting library for metabolic diversity, and
Identify clones containing inserts that carry genes that may be involved in the developmental pathway
In order to detect and identify the type I PKS gene,
A screening technique was developed with the present invention.

[0709]   1 Bacterial strains, plasmids and culture conditions   S. coelicolor ATCC1
01478, Streptomyces ambofaciens (S. ambofaci)
ens) NRRL 2420, Streptomyces lactamandurans (S.
Lactamandurans) ATCC 27382, Streptomyces re
S. rimosus ATCC 109610, Bacillus subtilis (B
． Subtilis) ATCC 6633 or Bacillus lichenifornis (B
． licheniformis) THE1856 (collection RPR)
Used as a source of DNA for PCR experiments. Streptomyces lividans
(S. lividans) TK24 used for shuttle cosmid POSI700
It is a host strain that

For production of suspensions of genomic DNA, protoplasts and spores, and transformation of S. lividans, Ho was used.
The standard protocol described in pwood et al. (1986) was followed.

As a host for cloning the PCR product, Escherichia coli (Escheri) was used.
chia coli) Top10 (INVITROGEN) was used, and E. coli Sur was used as a host for the shuttle cosmid pOS700I.
e (STRATAGENE) was used. Culture conditions of E. coli,
Plasmid preparation, digestion of the DNA and agarose gel electrophoresis were performed according to standard protocols (Sambrook et al., 1996).

[0712]   2. PCR primer   The primer pairs a1-a2 and b1-b2 are N. Bamas-Jacque
of the gene encoding PKSI, defined by a team of
For screening of soil libraries and of DNA from pure strains for research
Optimized for.

[0713]

[Table 1]

[0714]   Amplification conditions:   In the case of PKS I studies from pure strain DNA, the amplification mixture was
50-150 ng of genomic DNA, 200 μM dNTP, in a volume of 50 μl
5 mM MgCl_Two(Final), 7% DMSO, 1xAppligene buff
Each 0.4 μM of each primer and 2.5 U of Appligene
  It contained Taq polymerase. The amplification conditions used are as follows:
Denaturation at 95 ° C for 2 minutes, hybridization at 65 ° C for 1 minute, 1 at 72 ° C
Extension for 1 minute (first cycle), then the temperature is reduced to 58 ° C. for 30 cycles
(As described by K. Seow et al., 1997). The final extension step is 72 ° C for 10
Do for a minute.

In the case of PKSI studies from the DNA of the library, the PCR conditions were as described above for the a1-a2 pair using 100-500 ng cosmid extracted from a pool of 48 clones. Under the same conditions as.

For the b1-b2 primer pair, 500 ng of cosmid from a pool of 96 clones was used. The amplification mixture contained 200 μM dNTP, 2
． 5 mM MgCl ₂ (final), 7% DMSO, 1 × Quiagen buffer, 0.4 μM each primer and 2.5 U hot-st.
art) Taq polymerase (Qiagen). The amplification conditions used were as follows: denaturation at 95 ° C. for 2 minutes, followed by 30 cycles: denaturation at 95 ° C. for 1 minute + 65 ° C. (first cycle) and 62 ° C. (remaining cycles) for 1 minute high. Hybridization, extension at 72 ° C for 1 minute, final extension step at 72 ° C for 10 minutes.

Identification of positive clones from a pool of 48 or 96 clones is performed using replicas of the corresponding parental microplate on solid medium or using other standard replication methods.

[0718]   3 Subcloning and sequencing   PCR products of identified clones are sequenced according to the following protocol
. The fragment was purified on an agarose gel (gel extraction kit (Qiagen)) and
E. coli using the OPO TA cloning kit (Invitrogen)
E. coli) TOP 10 (Invitrogen).
The plasmid DNA of the subclone was cloned on Biobot (Qiagen).
Extracted by Lucari cell lysis, 0.025 μm VS membrane (Millipore)
Dialyze for 2 hours above. The sample was submitted to the ABI 377 96 sequencer (P
"Universal" and "Reverse" on erkin Elmer
Sequence with M13 primer.

[0719]   4) Results   Clarification and demonstration of the PCR primer   Degenerate two highly conserved regions of actinomycete type I PKS containing the enzyme active site
Targeted for amplification of homologous genes with primers. These two areas are
They correspond to the sequences PQQR (L) (L) LE and VE (A) HGTGT, respectively.
It

Strains that produce or do not produce macrolides, ie, Streptomyces
Kericolor (Streptomyces coelicolor), Streptomyces ambofaciensu (Streptomyces ambofac)
iens) (produces spiramycin) and Saccharopolyspora erythra (S
The primers (Table 8) were tested with the DNA of accharopolyspora erythraea (producing erythromycin). Regardless of the primers used, bands corresponding to the approximately 700 bp fragment and corresponding to the expected fragment length were obtained in all of these strains.

These results show that S. coelicol
or), the specificity of the primers a and b for the silent gene or for the PKS I gene of the producing strain.

Sequencing of the PCR products obtained with the a1-a2 primer pair has already been described as belonging to the biosynthetic pathway of plantenolide, the macrolide precursor of spiramycin (European patent application no. EP 0 791 656). ) Made it possible to identify the sequence of the KS gene from a S. ambofaciens strain (two sequences not listed; Stramb
9 and Stramb12 (see sequence listing)).

For S. erythraea, the screening method is based on 6-deoxyerythronolide B synthetase 1 (DEB
The sequence of KS (s) that is identical to the sequence of KS of module 1 already published in Genebank (accession number M63677) encoding S1)
acery17). Another sequence was identified that did not correlate with the erythromycin biosynthetic pathway. It is shown in SEQ ID NO: 32.

[0724]   Conclusion   Analysis of the presence of type I PKS-encoding genes from various microorganisms by PCR
Developed a method to do. Highly conserved type I keto-synthetase domain
The structure of the degenerate primer is biased towards GC for codon selection.
It has made it possible to generate a usage-based PCR method.

This approach has the potential to identify genes or clusters involved in the biosynthetic pathway of type I polyketides. Cloning of these genes allows the generation of populations that can later be used to construct polyketide hybrids. The same principles can be applied to other classes of antibiotics.

The results obtained here also indicate the presence of genes that can belong to the silent cluster (SEQ ID NOs: 30-32).

The existence of silent clusters is due to Streptomyces lividans (S. liv.
and their expression is induced by specific or polymorphic regulators (Horinouchi et al .; Umeyama et al.).
1996). These results show that the detection of genes belonging to the so-called silent pathway actually encodes an active enzyme that can direct the enzymatic steps required for the synthesis of the secondary metabolite, along with other specific enzymes of the pathway. Suggesting that

[0728]   Library screening   Using validated primer pairs from production strains,
Screening was performed under the conditions described in Section.

The size of the PCR product obtained from cosmid DNA extracted from a pool of 48 or 96 clones in the presence of the a1-a2 primer pair was approximately 700 bp. Therefore, this is consistent with the expected results.

The intensity of the bands obtained varied, but for each pool of target DNA:
There was only one amplified band.

Under these conditions, 8 groups of target DNAs corresponding to the 9 positive clones after deplication were detected.

Screening performed with the second primer pair b1-b2 gave less specific amplification results. Because of the number of attendants (with its 700bp band
This is because the satellite band was observed. Nevertheless, 9 groups of target DNA corresponding to 14 positive clones were detected after deplication starting from these positive clones. The DNA was extracted for the steps of sequencing and transformation of S. lividans.

[0733]   Analysis of the cosmid   Kosumi identified by PCR with the enzyme DraI recognizing AT-rich site
Digestion of cleaves releases fragments larger than 23 kb (Figure 22). This is the PCR
Suggests that the method preferentially targets soil DNA containing a high proportion of G + C
is doing. This result indicates that the primer used to select the codon (which is biased towards GC
This is due to the degeneracy of (biasing). The insert is a cosmid
23 kb except for one case (clone a9B12) as expected for
Larger size, which reflects some level of instability of the cosmid
Will be. In addition, of all the selected clones, two of them (G
S. F1 and GS. G11) only showed the same restriction profile, which
Indicates a low level of redundancy in the library.

Selected cosmids were transformed with Streptomyces lividans by transformation of protoplasts in the presence of PEG 1000.
(dans). The transformation efficiency was 1 μg of cosmid DNA used
The range is 30 to 1000 transformants per unit.

[0735]   Sequencing and phylogenetic analysis of the soil PKS I gene   The PCR method developed for pure strains was used for the cosmids of the library.
Used as described, 24 clones were thus identified.

A ˜700 bp PCR product from the DNA of the two pools (48 clones) and the 8 unique clones was cloned and sequenced after purification on agarose gel. This allowed the identification of 11 sequences.

Alignment of the deduced protein sequence of soil PKS I to other PKS I of various microorganisms (FIG. 24) shows the presence of a highly conserved region corresponding to the consensus region of the active site of β-ketoacyl synthetases. Is shown.

The “codon preference” method (Gribskov
Et al., 1984; Bibb et al., 1984) showed the presence of a strong bias in the use of G + C rich codons within a single reading frame. Proteins predicted according to this reading frame show strong similarity to known type I KS (Blast program). In particular, the similarity between the KS sequence from the soil and the KS sequence of the erythromycin cluster is about 53%.

After deplication of the pool and identification of the unique clone, the sequence of the PCR product obtained from this clone was identical to that of the pool, which indicated the reliability of the method used. Has proved.

Analysis of the sequences of the PCR products of the clones allowed the reliable identification of three different KSI genes. One of these sequences (SEQ ID NO: 34) has 98.7% similarity to the sequence of the other pool, indicating that they encode the same enzyme. There is. The remaining two sequences differ but show strong homology.

Soil D of the secondary metabolite biosynthetic pathway containing the gene encoding type I IKS
Cloning and identification in NA libraries is the first described in the present invention.

The high proportion of G + C in the soil sequence suggests that they may be derived from a genome with similar codon usage in the case of actinomycetes.

Although the data available from the literature are limited, the genes encoding type I PKS are in terms of their physical organization in the genome, size and number of modules contained within each gene. It is known that they are very diverse.

The presence of several domains derived from a single clone proves that they belong to an asymmetric (asymmetric) polyketide cluster. In one case,
It seems that the two clones form a contigum. Because they share the same sequence of the KS domain.

The size of the genetic region involved in PKSI synthesis ranges from a few kb of penicillin to about 120 kb of paramycin. Therefore, the size of the cosmid insert may not be sufficient for expression of most complex clusters.

The gene encoding PKS I, which can act as iteratively as PKS II and regulate the synthesis of aromatic polyketides, has already been described (Jae-Hyuk).
Et al., 1995). The study of soil PKS I clusters could provide further novelty in this field.

[0747]   5. Identification of 6 genes encoding polyketide synthetases   The screen of the cosmid library was screened according to the protocol described in this example.
Continuing the study, the present inventors found that the polyketide synthase-type polypeptide was
34071 bp containing several open reading frames that encode
A cosmid clone containing the insert was identified.

More particularly, the cosmids thus identified by screening the library were 6 open, encoding polyketide synthase polypeptides or very closely related polypeptides, non-ribosomal synthase peptides. Contains a reading frame.

The complete nucleotide sequence of the cosmid constitutes SEQ ID NO: 113 of the Sequence Listing. The DNA insert contained in SEQ ID NO: 113 constitutes the complementary nucleotide sequence (-strand) of the nucleotide sequence encoding the various polyketide synthases.

The nucleotide sequence of the DNA insert contained in the cosmid of Figure 36 containing the open reading frame (+ strand) encoding the polyketide synthase polypeptide is illustrated in Figure 37. The sequence constitutes sequence SEQ ID NO: 114 in the sequence listing.

Further, a detailed map of various open reading frames contained in the DNA insert of this cosmid is shown in FIG.

The characteristics of the nucleotide sequence containing the open reading frame contained in the DNA insert of this cosmid are detailed below.

[0753]   ORF1 array   The orf1 sequence has a partial open reading frame of 4615 nucleotides in length.
Including This sequence begins at nucleotide position 1 of SEQ ID NO: 114 and is
Sequence SEQ ID NO: 115, ending at position 4615 of Leotide.

SEQ ID NO: 115 encodes a 1537 amino acid ORF1 polypeptide,
This polypeptide constitutes SEQ ID NO: 121.

The polypeptide of SEQ ID NO: 121 is related to a non-ribosomal synthase peptide. This polypeptide is referred to in the Genbank database with the accession number "emb CACO1604.1".
baena) sp. It has 37% amino acid identity with 90 synthase peptides.

[0756]   ORF2 array   The orf2 nucleotide sequence is 8301 nucleotides long and is SEQ ID NO: 1.
14 nucleotides starting at nucleotide 4633 and ending at nucleotide 12933
Configure the column array number 116.

The ORF2 sequence encodes the 2766 amino acid ORF2 peptide, which polypeptide comprises SEQ ID NO: 122.

The polypeptide of SEQ ID NO: 122 has 41% amino acid sequence identity to the MtaD sequence of Stigmatella aurantia collated by the access number "gb AAF 19812.1" from the Genbank database. .

[0759]   The ORF2 polypeptide constitutes polyketide synthase.

[0760]   ORF3 array   The orf3 nucleotide sequence is 5292 nucleotides long and is SEQ ID NO: 1.
Make up 17. SEQ ID NO: 117 is nucleotide 1 of SEQ ID NO: 114.
It corresponds to the sequence starting at position 2936 and ending at nucleotide position 18227.

The nucleotide sequence SEQ ID NO: 117 encodes a 1763 amino acid ORF3 polyketide synthase polypeptide, which polypeptide is the sequence SEQ ID NO: 1 of the invention.
Make up 23.

The ORF3 polypeptide of SEQ ID NO: 123 is a Mta of Stigmatella aurantiaca which is collated from the Genbank database by the accession number “gb AAF 19810.1”.
It has 42% amino acid identity to the B sequence.

[0763]   ORF4 array   The orf4 nucleotide sequence is 6462 nucleotides long, and
Configure column number 118.

Nucleotide sequence SEQ ID NO: 118 corresponds to the sequence of nucleotide sequence SEQ ID NO: 114 starting at nucleotide position 18224 and ending at nucleotide position 24685.

The nucleotide sequence SEQ ID NO: 118 encodes a 2153 amino acid ORF4 polyketide synthase polypeptide, which polypeptide is SEQ ID NO: 1 of the invention.
Make up 24.

The ORF4 polypeptide of SEQ ID NO: 124 is identified as Sorangium •, collated by the access number “gb AAF62883.1” from the Genbank database.
It has 46% amino acid sequence identity to the epoD sequence of Sorangium cellulosum.

[0767]   ORF5 array   The orf5 nucleotide sequence is 5088 nucleotides long and
Configure column number 119.

SEQ ID NO: 119 is nucleotide 24 of the nucleotide sequence SEQ ID NO: 114.
It corresponds to the sequence starting at position 682 and ending at nucleotide 29769.

The nucleotide sequence SEQ ID NO: 119 encodes a 1695 amino acid ORF5 polyketide synthase polypeptide, which polypeptide is a SEQ ID NO: 1 sequence of the invention.
Make up 25.

The ORF5 polyketide synthase polypeptide of SEQ ID NO: 125 is Gen
Sorangium cellulosium collated by access number "gb AAF62883.1" from the bank database
um) epod sequence with 43% amino acid identity.

[0771]   ORF6 array   The orf6 nucleotide sequence is 4306 nucleotides long, and
Configure column number 120. The nucleotide sequence SEQ ID NO: 120 is SEQ ID NO: 1.
Start at nucleotide 29766 at position 14 and end at nucleotide 34071
Corresponds to an array.

SEQ ID NO: 120 is a polyketide synthase form of the 1434 amino acid ORF
It contains a partial open reading frame encoding 6 polypeptides, which constitutes SEQ ID NO: 126 of the invention.

The polypeptide of SEQ ID NO: 126 is 43 against the epoD sequence of Sorangium cellulosum matched by the access number "gb AAF62883.1" from the Genbank database.
It has a% amino acid identity.

[0774]   Example 15: Built-in BAC type shuttle probe in Streptomyces Construction   Built-in and mated BAC-type shuttle probes in Streptomyces Construction   15.1 Construction of vector pMBD-1   The vector BAC pMBD-1 was obtained according to the following steps.

Step 1 : Vector pOSVO10 was digested with the enzymes PsTI and BstZ171 to give a 6.3 kb nucleotide fragment.

Step 2 : The vector pDNR-1 was digested with the enzymes PstI and PvuII and 441
A 5 kb nucleotide fragment was obtained.

Step 3 : The 6.3 kb nucleotide fragment derived from the vector pOSV017 was fused by ligation with the 4.15 kb fragment derived from the vector pDNR-1 to obtain the vector pMBD-1 (see FIG. 30). As shown).

[0778]   15.2 Construction of vector pMBD-2   The vector pMBD-2 has a "φc31 int-Ωhyg" built-in box.
It is a BAC type vector contained.

Φc31 is a broad host-spectrum temperate phage with well-located adhesion sites (attP). The φc31 int fragment is the smallest fragment of actinophage φc31 capable of inducing the integration of the plasmid into the chromosome of Streptomyces lividans.

Ωhyg is a derivative of the Ωinterposon that can confer hygromycin resistance in E. coli and S. lividans.

BAC vectors containing the φc31 integration system are described in Sosio et al. (2000) and PCT Patent Application No. 99/6734 published December 29, 1999.

[0782]   The vector BAC pmBD-2 was constructed according to the following steps.

Step 1 : φc31i in E. coli multicopy plasmid
Construction of nt Ωhyg integration box First, the φc31int fragment is amplified from plasmid pOJ436 using the following primer pairs.

• Primer EVφc31I (SEQ ID NO: 109), which allows the introduction of an EcoRV site within the 5'end of the φc31 sequence and primer BIIφ.
c31F (SEQ ID NO: 110) (this is the BgL into the 3'end of the φc31 sequence
II site can be introduced).

The plasmid pHP described in Blondelet-Rouault (1997).
Digestion of 45 Ωhyg using the BamHI enzyme yielded the Ωhyg fragment.

The φc31 int-Ωhyg integration box was then cloned into the vector pMCS5 digested with the enzymes BglII and EcoRV.

[0787]   Step 2: Construction of vector pMBD-2   Fermentation of bacterial artificial chromosome pBAce3.6 described in Frengen et al. (1999).
Digested with elemental NheI and then treated with the enzyme Eco polymerase.

Next, the vector pMCS5 φc31 int-Ωhyg was digested with the enzymes SnaBI and EcoRV to recover the integration box.

[0789]   A detailed map of the vector pMBD2 is shown in FIG.

[0790]   15.3 Construction of vector pMBD-3   The vector pMBD-3 contains a BAC type integration containing the selectable marker Ωhyg.
It is a type (φc31 int) and mating type (OriT) vector.

[0791]   A map of the vector pMBD-3 and its construction method are shown in FIG.

Vector pMBD-3 was obtained by amplifying the OriT gene starting from plasmid pOJ436 using the primer pair of SEQ ID NO: 111 and SEQ ID NO: 112 containing a pacI restriction site.

The nucleotide fragment amplified using the primers SEQ ID NO: 111 and SEQ ID NO: 112 was cloned into the vector pMBD2 which was pre-digested with PacI enzyme. The scheme for the construction of vector pMBD-3 is shown in FIG.

[0794]   15.4 Construction of vector pMBD-4   A detailed map of the vector pMBD-4 is shown in FIG.

The vector pMBD-4 was cloned into the vector pCYTAC2 with φc31 int-Ω.
Obtained by cloning the hyg integration box.

[0796]   15.5 Construction of vector pMBD-5   The scheme for the construction of vector pMBD-5 is shown in FIG.

Vector pMBD-5 is the two lox of vector pMBD-1 shown in FIG.
The nucleotide fragment contained between the P sites was constructed by recombination at the loxP site contained within the BAC vector called pBTP3. Plasmid pBT
A detailed map of P3 is shown in FIG.

[0798]   15.6 Construction of vector pMBD-6   The nucleotide fragment contained within the two loxP sites of the vector pMBD-1
By recombination within the loxP site of the BAC pBeloBac11 vector
, Vector pMBD-6 was constructed (as described in FIG. 35).

[0799]

[Table 2]

[0800]

[Table 3]

[0801]

[Table 4]

[0802]

[Table 5]

[0803]

[Table 6]

[0804]

[Table 7]

[0805]

[Table 8]

[0806]

[Table 9]

[0807]     (References)

[0808]

[Table 10]

[Sequence list]

[Brief description of drawings]

FIG. 1 shows a scheme of various cell lysis steps performed according to the protocols 1, 2, 3n, 4a, 4b, 5a and 5b described in Example 1.

FIG. 2 shows that 0 mg of DNA extracted from 300 mg of soil No. 3 (St Andre coast) after various cell lysis treatments (protocols 1-5, see FIG. 1).
． Electrophoresis on an 8% agarose gel is shown. M: Lambda phage molecular weight marker.

FIG. 3 shows the proportions of various genera of actinomycetes cultured after treatments 1-5 (see FIG. 1). Cfu (colony forming units) values were measured on media selective for this group of bacteria. A total of about 400 colonies were analyzed.

FIG. 4 shows Hin added to soil at various concentrations before (G) or after (G ^* ) milling.
6 shows recovery of lambda phage DNA digested with dIII. Treatment T (heat shock)
And S (sonication) are additional cell lysis treatments. The quantification was performed by analysis on a phosphoimager after dot blot hybridization. Samples of each soil were used for each concentration of lambda phage added. The characteristics of the soil are shown in Table 1. Samples corresponding to the added 10-15 μg pf DNA were untreated.

FIG. 5 shows PCR amplification of DNA extracted from soil number 3 according to Protocols 1, 2, 3, 5a and 5b. Resident Streptosporangium species (Strep
tosporangium spp. ) For targeting
S 122 and FGPS 350 (Table 2) were used. The extracted DNA was used undiluted or at 10-fold and 100-fold dilutions. M: 123 bp molecular weight marker (Gibco BRL), C: amplification control without DNA.

6 is a diagram of S. lividans OS48.
． DNA extracted after inoculating the soil with various concentrations of spores or mycelium of No. 3
Indicates the amount of The amount of mycelium added to the soil corresponds to the number of spores inoculated into the germination medium. About 50% of the spores germinated and the number of cells or genome contained in the germinated spore hyphae was not determined. Therefore, the amount of inoculated spores and mycelia cannot be directly compared. The extraction protocol was performed according to Protocol 6 (see Materials and Methods section). The symbol (') indicates the RN in the extraction buffer.
It indicates that A was included. The target DNA was amplified by PCR with primers FGPS 516 and FGPS 517 and the quantification was performed using the probe FGPS 5
Performed on a phosphoimager after dot blot hybridization using 18. Samples of each soil were used for each concentration of hyphae or spores. The characteristics of the soil are shown in Table 1.

FIG. 7-a) FIG. 7 represents a phylogenetic tree obtained by the Neighbour Joining algorithm in which the 16S rDNA sequence contained in the soil DNA library is arranged with respect to a culture reference bacterium. Gray display: sequences obtained from a pool of clones of the library. The bootstrap value after 100 iterations of resampling is shown at the aggregation point. The scale line shows the number of substitutions per site. The access numbers for the sequences in the Genbank database are shown in parentheses.

FIG. 7-b) FIG. 7 represents a phylogenetic tree obtained by the Neighbour Joining algorithm in which the 16S rDNA sequence contained in the soil DNA library is arranged with respect to a culture reference bacterium. Gray display: sequences obtained from a pool of clones of the library. The bootstrap value after 100 iterations of resampling is shown at the aggregation point. The scale line shows the number of substitutions per site. The access numbers for the sequences in the Genbank database are shown in parentheses.

[Figure 8]   FIG. 8 represents the scheme of the vector pOSint1.

[Figure 9]   FIG. 9 represents the scheme of the vector pWED1.

FIG. 10 represents the scheme of the vector pWE15 (ATCC No. 37503).

FIG. 11   FIG. 11 represents the scheme of the vector pOS700I.

[Fig. 12]   FIG. 12 represents the scheme of the vector pOSV010.

FIG. 13 represents a fragment containing the “cos” site inserted into plasmid pOSV010 during construction of vector pOSV303.

FIG. 14   FIG. 14 represents the scheme of the vector pOSV303.

FIG. 15   FIG. 15 represents the scheme of the vector pE116.

FIG. 16   FIG. 16 represents the scheme of the vector pOS700R.

FIG. 17   FIG. 17 represents the scheme of the vector pOSV001.

FIG. 18   FIG. 18 represents the scheme of the vector pOSV002.

FIG. 19   FIG. 19 represents the scheme of the vector pOSV014.

FIG. 20   FIG. 20 represents the scheme of the vector pBAC11.

FIG. 21   FIG. 21 represents the scheme of the vector pOSV403.

FIG. 22 shows an electrophoretic gel of the DNA of the library after digesting positive clones of the library screened with PKS-1 oligonucleotides with the enzymes BamHI and DraI.

Figure 23 shows the production of puromycin by S. lividans recombinants as compared to the production of S. alboniger wild type strains.

FIG. 24 shows an alignment of soil PKS with the conserved active sites of other PKSs. The display content for each peptide is shown. The beta-ketoacyl synthase domain has the GCG PILEUP program (Wisconsin P
ackage Version 9.1, Genetics Computer
Aligned using Group, Madison, Wisc).

FIG. 25   FIG. 25 shows the construction of an integrative mating cosmid.

FIG. 26   FIG. 26 shows the construction of a built-in mating BAC.

FIG. 27   FIG. 27 shows a scheme for the construction of vector pOSV308.

FIG. 28   FIG. 28 shows a scheme for the construction of vector pOSV306.

FIG. 29   Figure 29 shows the scheme for the vector pOSV307.

FIG. 30   FIG. 30 shows a scheme for the construction of vector PMBD-1.

FIG. 31. Detailed map of plasmid pMBD-2 and vector pMBD-3.
A scheme for the construction of is shown.

FIG. 32   Figure 32 shows a detailed map of plasmid pMBD-4.

FIG. 33 shows a scheme for the construction of plasmid pMBD-5 from plasmid pMBD-1.

FIG. 34   Figure 34 shows a detailed map of the vector pBTP-3.

FIG. 35 shows a scheme for the construction of vector pMBD-6 from vector pMBD-1.

FIG. 36 shows a map of cosmid a26G1 whose DNA insert contains an open reading frame encoding several polyketide synthases.

FIG. 37 is a scheme depicting the DNA insert (+ strand) of cosmid a26G1 arranged with various reading frames encoding several polyketide synthases.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 33/02 A61P 35/00 4C086 35/00 37/06 4C087 37/06 C07K 16/40 4H045 C07K 16 / 40 C12N 1/15 C12N 1/15 1/19 1/19 1/21 1/21 9/10 9/10 C12P 1/00 Z C12P 1/00 C12Q 1/02 C12Q 1/02 1/68 A 1 / 68 G01N 33/53 M G01N 33/53 33/566 33/566 33/573 A 33/573 C12R 1: 465 // (C12N 1/21 C12N 15/00 ZNAA C12R 1: 465) (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE, TR), OA (BF, BJ, CF, CG) , CI, CM, GA, GN, G W, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY) , KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CR, CU , CZ, DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, RU, SD, SE, SG , SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Simone, Pascal France, F-69100, Beuil-Burbanne, Liu-Pierre-Boyan, 55 (72) Inventor Kurtwa, Sophie France, E-94220, Charenton-le-France Pont, Liuux-de-Paris, 165 (72) Inventor Caperano, Carmela France, F-94120, Fountain sue Bois, Liuux-de-Nouilly, 16 (72) Inventor Frank, Francois France, Ehu -91120, Palaiseau, Boulevard d'Auxerre, 76 (72) Inventor Renal, Alain, France, EF-91440, Biul-Sheur-Ibet, Avenyu-de-Teiyul, 52 (72) Invention Persons Bar, Maria Venezuela, Merida, Merida Estado, Apartament 42, Piso 4, Edificio 10, Lesquemar Cardinal Quintero, Avenir・ Cardenal Quintella (72) Inventor Susono, Gunaday France, F-75002, Paris, Riu Saint-Sabourg, 16 (72) Inventor Téhuil, Karine France, F-91400, Orsay, Boulevard de Ubreuil , 39/41 (72) Inventor Frosteguard, Asa Norway, N-1450, Nesodotangen, Fretebay Skogosviy 7F Term (reference) 4B024 AA01 AA11 BA10 BA67 CA03 CA09 CA20 DA06 DA08 DA11 DA12 EA06 FA15 GA16 HA03 HA12 4B050 CC01 CC03 DD01 EE10 LL01 4B063 QA01 QA12 QA18 QQ06 QQ07 QQ13 QQ15 QQ19 QQ20 QQ98 QR56 QR62 QR75 QR76 QR80 QS24 QS25 4B064 AE41 AF54 CA05 CA06 CA19 CA24 A02 A44A02 A44A02 A44A02 NA14 ZB32 ZB35 ZB37 4C087 AA01 AA03 BC01 BC15 BC30 CA11 CA12 CA13 CA22 MA01 NA14 ZB32 ZB35 ZB37 4H045 AA11 AA30 CA11 DA75 EA50 FA71

Claims (81)

[Claims] 1. A method of producing a population of nucleic acids from a soil sample containing organisms, comprising the steps in the following order: I (a) fine particles by grinding a pre-dried or pre-dehydrated soil sample. And then suspending the microparticles in a liquid buffer medium, (b) extracting nucleic acids present in the microparticles, (c) passing a solution containing the nucleic acids over a molecular sieve, and then
A process comprising the steps of recovering an elution fraction rich in nucleic acid, passing the elution fraction rich in nucleic acid onto an anion exchange chromatography carrier, and then recovering the elution fraction containing the purified nucleic acid. Method. 2. A method for producing a population of nucleic acids from an environmental sample containing organisms, the steps in the following order: II (i) a suspension by dispersing the environmental sample in a liquid medium. And then homogenizing the suspension by gentle agitation, (ii) the organism and other minerals and / or other of the homogeneous suspension obtained in step (i).
Alternatively, a step of separating the organic component and the organic component by centrifugation on a density gradient, (iii) cell lysing the organism separated in step (ii), and extracting the nucleic acid, (iv) on a cesium chloride gradient A production method comprising a step of purifying the nucleic acid. 3. The process according to claim 1, wherein step I- (a) is followed by an additional step of treating the microparticles suspended in the liquid buffer by sonication. 4. After step I- (a), the following additional steps are carried out: a step of sonicating the fine particles suspended in a liquid buffer; a lysozyme and an acromo after sonication. The method according to claim 1, wherein the step of incubating the suspension at 37 ° C. in the presence of peptidase, the step of adding SDS, and the step of recovering the nucleic acid follow. 5. After step I- (a), the following additional steps are carried out: homogenizing the microparticles by vigorous mixing (vortexing) followed by simple stirring. Freezing the homogenous suspension and then thawing; after thawing, treating the suspension by sonication; after sonication, the suspension in the presence of lysozyme and achromopeptidase. The method according to claim 1, wherein the step of incubating the suspension at 37 ° C. is followed by the step of adding SDS. 6. The production method according to claim 1, wherein the nucleic acid is a DNA molecule. 7. A method for producing a population of recombinant vectors, which comprises inserting the nucleic acid obtained by the method according to any one of claims 1 to 6 into a cloning and / or expression vector. 8. The production method according to claim 7, wherein the nucleic acids are separated according to their size before inserting the nucleic acids into a cloning and / or expression vector. 9. The method according to claim 7, wherein the average size of the nucleic acid is made substantially uniform by physical disruption prior to the cloning and / or insertion of the nucleic acid into the expression vector. 10. The method according to claim 7, wherein the cloning and / or expression vector is of the fasmid type. 11. The production method according to claim 7, wherein the cloning and / or expression vector is of the cosmid type. 12. The method according to claim 11, which is a cosmid that is replicative in E. coli and integrative in Streptomyces. 13. The method according to claim 12, which is cosmid pOS700I. 14. That is, Streptomyces.
The method according to claim 1, which is a cosmid that is a joint type and an embedded type in (1). 15. The cosmids are cosmids pOSV303 and pOSV306.
15. The manufacturing method according to claim 14, which is selected from pOSV307 and pOSV307. 16. The method according to claim 11, wherein the cosmid is a replication type cosmid in both E. coli and Streptomyces. 17. The method according to claim 16, which is cosmid pOS700R. 18. Streptomyces (S), which is replicative in E. coli and Streptomyces.
The production method according to claim 11, which is a cosmid that is a zygote type in treptomyces). 19. The method according to claim 7, wherein the cloning and / or expression vector is of the BAC type. 20. That is Streptomyces.
20. The method according to claim 19, which is a BAC vector that is integrative or conjugative in (1). 21. The BAC vectors pOSV403 and pMBD.
-1, pMBD-2, pMBD-3, pMBD-4, pMBD-5 and pMB
The manufacturing method according to claim 20, which is selected from D-6. 22. A method of producing a recombinant cloning and / or expression vector, wherein the step of inserting the nucleic acid into the cloning and / or expression vector is selected using an appropriate restriction endonuclease. Cleaving the cloning and / or expression vector at the cloning site, adding a first homopolymer nucleic acid at the free 3'end of the open vector, releasing the nucleic acid from the population to be inserted into the vector At the 3'end, the first
Adding a second homopolymeric nucleic acid having a sequence complementary to the homopolymeric nucleic acid, by hybridizing the first and second homopolymeric nucleic acids having sequences complementary to each other to obtain a nucleic acid of the vector and the population A method of assembling a nucleic acid; and a step of closing the vector by ligation. 23. The first homopolymeric nucleic acid is poly (A) or poly (T)
The second homopolymeric nucleic acid is of a poly (T) or poly (A) sequence,
The manufacturing method according to claim 22. 24. The size of the inserted nucleic acid is at least 100 kilobases,
The method for producing a recombinant vector according to claim 22 or 23, which is preferably at least 200 kilobases. 25. The production method according to any one of claims 22 to 24, wherein the nucleic acid to be inserted is contained in a population of nucleic acids obtained by the production method according to any one of claims 1 to 6. . 26. A method for producing a recombinant cloning and / or expression vector, wherein the step of inserting a nucleic acid into the cloning and / or expression vector comprises: removing overhanging 3 ′ sequences and filling in overhanging 5 ′ sequences. Creating blunt ends on the ends of the nucleic acids of the population by cleaving, cleaving the cloning and / or expression vector at a selected cloning site using a suitable restriction endonuclease. A step of creating a blunt end at the end of the vector nucleic acid by removing the overhanging 3'sequence and filling in the overhanging 5'sequence, and then dephosphorylating the 5'end, -adding a complementary oligonucleotide adapter A step of: -inserting the nucleic acids of the population into the vector by ligation Law. 27. The size of the inserted nucleic acid is at least 100 kilobases,
27. The method for producing a recombinant vector according to claim 26, which is preferably at least 200 kilobases. 28. The method according to claim 26 or 27, wherein the nucleic acid to be inserted is contained in the population of nucleic acids obtained by the method according to any one of claims 1 to 6. 29. The nucleic acid, without being treated with one or more restriction endonucleases prior to inserting the nucleic acid into the cloning and / or expression vector,
The manufacturing method according to any one of claims 22 to 28, which is inserted as it is obtained. 30. A population of nucleic acids consisting of the nucleic acids obtained by the method according to any one of claims 1 to 6. 31. A nucleic acid contained in the population of nucleic acids according to claim 30. 32. The nucleic acid of claim 31, comprising a nucleotide sequence encoding at least one operon or part of an operon. 33. The nucleic acid of claim 32, wherein the operon encodes all or part of a metabolic pathway. 34. The nucleic acid of claim 33, wherein the metabolic pathway is the polyketide synthetic pathway. 35. The nucleic acid according to claim 34, which is selected from the polynucleotides comprising SEQ ID NOs: 30-44 and SEQ ID NOs: 115-120. 36. The nucleic acid of claim 31, which comprises the entire nucleotide sequence encoding the polypeptide. 37. The nucleic acid according to any one of claims 31 to 36, which is derived from a prokaryote. 38. The nucleic acid according to claim 37, which is derived from a bacterium or a virus. 39. The nucleic acid according to any one of claims 31 to 33 and 36, which is derived from a eukaryote. 40. The nucleic acid according to claim 39, which is derived from a fungus, yeast, plant or animal. 41. The following recombinant vector: a) a vector containing the nucleic acid according to any one of claims 35 to 40, b) obtained by the production method according to any one of claims 22 to 25 and 29. A recombinant vector, c) selected from the vectors obtained by the production method according to any one of claims 26 to 29. 42. A vector which is the cosmid pOS700I. 43. A vector which is the cosmid pOSV303. 44. A vector which is the cosmid pOSV306. 45. A vector which is the cosmid pOSV307. 46. A vector which is the cosmid pOS700R. 47. A vector which is the BAC vector pOSV403. 48. A vector which is the vector pMBD-1. 49. A vector which is the vector pMBD-2. 50. A vector which is the vector pMBD-3. 51. A vector which is the vector pMBD-4. 52. A vector, which is the vector pMBD-5. 53. A vector which is the vector pMBD-6. 54. A population of recombinant vectors obtained by the method according to any one of claims 7 to 21, 25 and 28. 55. A recombinant cloning and / or expression vector, characterized in that it is comprised in the population of recombinant vectors according to claim 54. 56. A recombinant host cell comprising the nucleic acid of any one of claims 31-40 or the recombinant vector of claim 55. 57. The recombinant host cell of claim 56, which is a prokaryotic or eukaryotic cell. 58. The recombinant host cell of claim 57, which is a bacterium. 59. E. coli and Streptomyces (St)
59. The recombinant host cell according to claim 58, which is a bacterium selected from reptomyces). 60. The recombinant host cell of claim 58, which is a yeast or filamentous fungus. 61. A population of recombinant host cells, each host cell of the population comprising a nucleic acid from the population of nucleic acids of claim 30. 62. A population of recombinant host cells, each host cell of the population comprising the recombinant vector of claim 41 or 55. 63. A method for detecting a nucleic acid of a given nucleotide sequence or a nucleic acid of a nucleotide sequence structurally similar to a given nucleotide sequence in a population of recombinant host cells according to claim 61 or 62. Contacting the population of recombinant host cells with a pair of primers that hybridize to its given nucleotide sequence, or a pair of primers that hybridize to a nucleotide sequence structurally similar to the given nucleotide sequence. A step of: performing at least three amplification cycles; detecting an amplified nucleic acid; 64. A method for detecting a nucleic acid of a given nucleotide sequence or a nucleic acid of a nucleotide sequence structurally similar to a given nucleotide sequence in a population of recombinant host cells according to claim 61 or 62. Contacting a population of recombinant host cells with a probe that hybridizes to its given nucleotide sequence, or a probe that hybridizes to a nucleotide sequence that is structurally similar to its given nucleotide sequence, A method comprising the step of detecting hybrids that may form between the probe and the nucleic acid contained within the vector of the population. 65. One within the population of recombinant host cells according to claim 61 or 62.
A method for identifying the production of a compound of interest by a recombinant host cell as described above, comprising culturing the recombinant host cells of said population in a suitable medium, one or more cultured recombinant cells Detecting the compound of interest in the culture supernatant or the cell lysate of. 66. A method for selecting a recombinant host cell producing a compound of interest in the population of recombinant host cells according to claim 61 or 62, comprising: Culturing in a suitable medium, detecting the compound of interest in the culture supernatant or cell lysate of the cultured one or more recombinant host cells, the recombinant host cell producing the compound of interest A method comprising the step of selecting. 67. A step of culturing a recombinant host cell selected by the method according to claim 66, a step of recovering a compound produced by the recombinant host cell, and purifying it when appropriate. A process for producing a compound of interest, characterized in that 68. A compound of interest, obtained by the process according to claim 67. 69. The compound of claim 68, which is a polyketide. 70. A polyketide produced by expressing at least one nucleotide sequence comprising a sequence selected from SEQ ID NOs: 30 to 44 and SEQ ID NOs: 115 to 120. 71. A composition comprising the polyketide of claim 69 or 70. 72. A pharmaceutical composition comprising a pharmacologically active amount of the polyketide of claim 69 or 70 and a pharmaceutically acceptable vehicle. 73. A method for measuring the diversity of nucleic acids contained in a population of nucleic acids, in particular a population of nucleic acids from an environmental sample, preferably a soil sample, comprising: Contacting with a pair of oligonucleotide primers that hybridize to any sequence of bacterial 16S ribosomal DNA, performing at least three amplification cycles, one oligonucleotide probe or multiple oligonucleotide probes (each probe is , 16S ribosomal DN common to the genus, bacterium, order, subclass or genus
Hybridizing specifically to the sequence A)), and detecting the amplified nucleic acid, if appropriate, the results from the detection step are used for the nucleic acid of known sequence constituting the calibration range. A method comprising a step of comparing with a detection result when a probe or a plurality of the probes described above is used. 74. The method of claim 73, wherein the pair of primers that hybridize to any sequence of bacterial 16S ribosomal DNA consists of primer FGPS 612 (SEQ ID NO: 12) and primer FGPS 669 (SEQ ID NO: 13). 75. The method of claim 73, wherein the pair of primers that hybridize to any sequence of bacterial 16S ribosomal DNA consists of primer 63f (SEQ ID NO: 22) and primer 1387r (SEQ ID NO: 23). 76. At least 9 relative to SEQ ID NO: 60 to SEQ ID NO: 106
A nucleic acid comprising a 16S rDNA nucleotide sequence selected from sequences having 9% nucleotide identity. 77. SEQ ID NO: 33 to SEQ ID NO: 44 and SEQ ID NO: 30 to
A step of producing a recombinant host cell containing a nucleic acid encoding a type I polyketide synthase containing a nucleotide sequence selected from SEQ ID NO: 32 and SEQ ID NO: 115 to SEQ ID NO: 120, culturing the recombinant host cell in a suitable medium The method for producing a type I polyketide synthase, comprising the steps of: collecting the type I polyketide synthase from the culture supernatant or the cell lysate, and purifying the type I polyketide synthase if appropriate. 78. A polyketide synthase comprising an amino acid sequence selected from SEQ ID NOs: 45 to 59 and SEQ ID NOs: 121 to 126. 79. An antibody against the polyketide synthase of claim 78. 80. A method for detecting a type I polyketide synthase or a peptide fragment of this enzyme in a sample, comprising the steps of: a) contacting the antibody of claim 79 with the test sample; b) forming A potential antigen / antibody complex. 81. I) in a sample, comprising a) the antibody of claim 79, and b) where appropriate, the reagents necessary to detect possible antigen / antibody complexes that have formed. A kit for detecting type polyketide synthase.