FR2489363A1 - Hybrid plasmid(s) contg. genes for nitrogen fixation - to introduce nitrogen fixing ability into yeast cells for prodn. of foodstuffs - Google Patents

Abstract

Hybrid plasmids contg. (a) a DNA fragment of a yeast 2 micrometre plasmid, (b) a DNA segment contg. a yeast gene, (c) a bacterial plasmid DNA fragment, and (d) a DNA fragment contg. the genes responsible for fixing nitrogen, are new. Pref. the DNA fragment (a) is a fragment of the restriction between the Eco R1(1) and the Hind III (3) sites, as defined by Guerineau in Viruses and Plasmids in Fungi, Marcel Dekker, N.Y. 1979, ed. by P.A. Lemke. The yeast gene (b) is pref. URA3+ gene, the DNA fragments being adjacent to form a 'DNA yeast block.' The DNA fragment (c) pref. carries a lambda or lambdoid phage cos site to facilitate cloning, such as pHc 79 described by Collins, Methods in Enzymology, 68, 309, 1979. The DNA fragment carrying nitrogen-fixing genes is pref. derived from Klebsiella pneumoniae. Also claimed are cells and microorganisms contg. the hybrid plasmids. The plasmids may be used to introduce nitrogen-fixing genes into eucaryotic cells, esp. yeasts, their culture enabling foodstuffs to be produced without using nitrogenous fertilisers.

Description

 The present invention relates to "hybrid plasmids useful for the introduction of nif genes into prokaryotic or eukaryotic cells which lack them, in particular bacteria and yeasts.

 The nif genes ("nitrogen fixation") are the genes carrying the genetic information for the fixation of atmospheric nitrogen.

 The genetic information for the fixation of atmospheric nitrogen is only found in prokaryotic organisms (bacteria and cyanophyceae).

However, this information conditions the rest of life on earth since other living organisms (plants, animals) have an absolute need for combined nitrogen.

 The development of intensive agriculture could only be achieved by the massive supply of synthetic nitrogen fertilizers and it is clear today that the introduction into plants of the capacity to use atmospheric nitrogen would have considerable economic consequences.

 The present invention constitutes a first approach to the solution of this problem by providing plasmids allowing the introduction of the nif genes into a eukaryotic cell.

 I1 is a hybrid plasmid characterized in that it comprises at least - a DNA fragment of a yeast plasmid zende, - a DNA segment containing a yeast gene, - a DNA fragment d 'a bacterial plasmid, and - a DNA fragment containing the nif genes.

 In a preferred embodiment of these plasmids, the DNA fragment of plasmid 2; mess the restriction fragment R1 H3 of 2.12 Kb of the B form of the plasmid 2um.

 This fragment allows better amplification of the hybrid plasmid in yeast.

In the context of the present description, the abbreviations 1, Hind III "and" Eco R1 "will sometimes be used, when no ambiguity is permitted, to denote the restriction endonucleases Hind III and Eco R1, and the terms" site Hind III "or Eco R1 site" to designate the Hind III restriction sites or
Eco R1.

 The nomenclature of restriction sites for plasmid 2 will, unless otherwise indicated, refer to the restriction maps appearing in: Guerineau M.

Plasmid DNA in Yeast, in Lemke, P.A. (Ed), Viruses and Plasmids in Fungi, Marcel Dekker, New York, 1979.

 Thus, the R1 H3 restriction fragment is a restriction fragment between the Eco R1 site (1) and the Hind III site (3) as defined in the previous document.

 The DNA fragment of a bacterial plasmid will preferably carry a cos site of phage lambda or lambdoid which facilitates cloning as will be described below, but, for the implementation of the plasmid, the presence of a cos site is not essential.

The term “cos site” is understood to mean, in the case of phage lambda, the domain comprising the cohesive agglutinating ends. Thus, among the bacterial plasmids which can be fragmented to be introduced into a plasmid according to the invention, mention should be made of the cosmid (plasmid comprising a cos site) pHC 79 described in COLLINS J., Methods in
Enzymol., 68, 309, 1979 and particularly the Hind III Eco R1 restriction fragment of 6.5 Kb comprising the cos site.

 The DNA fragment carrying the nif genes preferably comes from Klebsiella pneumoniae.

In this bacteria, these genes are carried by two fragments of DNA, the ends of which are provided with nucleotide sequences recognized by the restriction enzyme Hind III. These fragments are of the order of 16 and 25 Kb, these are the 14 nif genes organized into 7 transcription units which have been identified.

 Finally, in the preferred embodiment of the plasmids according to the invention, the yeast gene is the URA3 gene and the yeast DNA fragments are contiguous in order to form a yeast DNA block ".

 The present invention also relates to microorganisms and eukaryotic cells, in particular yeasts, comprising a plasmid as defined above. Among these microorganisms, mention should be made more particularly of bacteria and especially yeasts.

 The examples below are intended to illustrate certain characteristics of the plasmids according to the present invention but cannot in any way limit it.

 The use of restriction enzymes and ligases is known to those skilled in the art and in order to simplify the description, unless otherwise indicated, the enzymes in question will be used in accordance with the manufacturer's instructions. These enzymes are sold in particular by the company BIOLABS, the company MILES LABORATORIES INC.

and the company BOEHRINGER.

Example 1
Preparation of plasmid pG 63
A - The plasmid pBR 322 (used by BETHESDA) is used as the starting plasmid
RESEARCH LABORATORY, INC., Rockville, Maryland.)
This plasmid is digested simultaneously with two restriction enzymes: endo Eco R1 and Hind III, according to the conditions described by the manufacturer (BIOLABS). The plasmid is cut into two fragments, one of 31 base pairs and the other of 4331 base pairs (SUTCLIFFE JC Nucleic Acids Research 1978, 5, 2721-2728). The two fragments are separated by electrophoresis in 1% agarose gel and the large fragment is recovered by the freeze-thaw technique (COLBERE-GARRAPIN F.,
CHOUSTERMAN S., HORODNICEANU F., KOURILSKY P., CARMIN A. Proc. Nat. Acad. Sci. USA 1979, 76, 3755-3759.)
B - The 2 μm plasmid is extracted from the yeast strain T8 (GUERINEAU M., SLONIMSKI PP,
AVNER PR Biochemical Biophys. Res. Com. 1974 ', 61, 462-469).

I1 is also digested simultaneously by the two restriction enzymes endo Eco R1 and endo
Hind III (BIOLABS).

 The restriction map of plasmid 2mm shows that there are two forms A and B of the 2pm plasmid.

The Eco R1 (1) Hind III (3) (RI H3) fragment of form B with a molecular weight of 2.12 Xb is purified by electrophoresis in agarose gel t 1% and it is collected from the gel by freezing-thawing technique already described.

 C - The fragment pBR 322 and the 2.12 Kb yeast fragment mentioned above are mixed and ligated with DNA ligase extracted from bacteriophage T4 under the conditions indicated by the manufacturer (BIOLABS).

Since the DNA fragment of the plasmid pBR 322 carries the Ampr gene, the ligation mixture is used to transform a strain of E. coli ampicillin sensitive and bacteria that have become ampicillin resistant are selected. The extracted plasmid, called pG 6 and described in French patent application no. 80 18755 filed on the same day as the present application in the name of
National Center for Scientific Research (CNRS) and entitled "New hybrid plasmids and microorganisms containing them", contains the RI H3 piece of 2 pm of yeast inserted in the RI H3 sites of pBR 322.

D - The URA 3 gene is obtained by digestion with
Hind III of plasmid pBR 322 - URA 3 clone 6 (BACH ML,
LACROUTE F., BOTSTEIN D., Proc. Nat. Acad. Sci. USA 1979, 76, 386-390) and the 1.1 Kb fragment is purified and harvested by the techniques described above. I1 is mixed with pG 6 previously digested with Hind III and then ligated as previously described. The mixture is used to transform an E. coli pyr F bacterium. The transformants capable of multiplying in the absence of uracil are selected.

représente 1'ADN du gène URA 3 - la partie

représente 1'ADN du plasmide pBR 322 - la partie

représente le fragment d'ADN du
plasmide 2Wm - la partie vs4s représente une séquence répétée
inverse du plasmide 2um. From these transformants, a new chimeric plasmid pG 63 is extracted, which is described in FIG. 1.
In this figure 1 J - the part

represents the DNA of the URA 3 gene - the part

represents the DNA of the plasmid pBR 322 - the part

represents the DNA fragment of the
2Wm plasmid - the vs4s part represents a repeated sequence
reverse of plasmid 2um.

 The restriction sites are noted as follows - R restriction site Eco R1 - H restriction site Hind III - P restriction site Pst - Hpa restriction site Hpa - B restriction site Bam H1.

This plasmid therefore contains a yeast DNA block of 3.3 Kb located between a restriction site
Eco R1 and Hind III and consisting of the 2.12 Xb DNA fragment of the yeast 2imide plasmid and a 1.1b DNA fragment carrying the URA 3 gene. The bacterial fragment which completes the plasmid consists of the DNA fragment of pBR 322 comprising 4331 base pairs.

 It should be noted that this plasmid has a Hind III site in the yeast DNA block which was used to insert the URA 3 gene.

 In order to obtain a vector plasmid of interest, it should have a maximum of unique restriction site, in particular a unique Hind III restriction site which corresponds to one of the most commonly used restriction enzymes.

This is why we must delete this Hind III site.

Example 2
Preparation of the plasmid pG 63 11
The Hind III site located between the 2 μm yeast part and the URA 3 gene of pG 63 was deleted as follows: the plasmid pG 63, deposited at the CNCM (National Collection of Microbial Cultures at the Pasteur I Institute in Paris) under nO I-130, was partially digested with Hind III. The full length linear molecules are separated and purified by agarose gel electrophoresis. These molecules are then treated with the endonuclease SI specific for single-strand DNA (BIOLABS) under the conditions described by the manufacturer, except the temperature which is 200C.

 After treatment, the DNA is precipitated and ligated with DNA ligase from phage T4 (BIOLABS) I at high concentration, which makes it possible to ligate blunt ends without cohesive ends.

 As before, after ligation, the ligation mixture is used to transform an E. coli ampicillin-sensitive strain and the resistant ampicillin bacteria which necessarily contain the plasmid are selected.

 After analysis of the plasmids which are extracted and which are analyzed by restriction, the plasmid pG 63 11 shown in FIG. 2 is obtained.

 This plasmid has the same overall structure as the plasmid pG 63 described above, but the Hind III restriction site located between the yeast part 2 and the URA 3 gene has been deleted.

The structure of this plasmid is particularly interesting. Indeed,
1) it has several unique restriction sites: Eco R1, Hind III, Bam H1 and Hp al
2) the Eco R1 Hind III restriction fragment consists only of yeast DNA, that is to say that a yeast block has thus been constructed which can easily be transposed into other vectors
3) resistance I tetracycline is maintained and the incorporation of a foreign DNA at the Bam H1 site leads to the inactivation of the gene and therefore gives a sensitive tetracyline phenotype; resistance I to ampicillin is also maintained but, however, there are three Ps tI sites in this vector.

Example 3
Preparation of the cosmid pHCG 3
From the plasmid pG 63 11, the yeast block is extracted by digestion of the plasmid with Hind III and
Eco R1 as described in Example 1-B.

Likewise, the cosmid pHC 79 described by COLLINS J. Methods in
Enzymol., 68, 309, 1979 by Hind III and Eco R1 by electrophoresis on agarose gel, the Eco R1 Hind III restriction fragment of 6.5 Kb is recovered, comprising the sitecos.

 By ligation of the two DNA fragments obtained, as described in Example 1-C, the cosmid pHCG 3 is obtained having the structure shown in FIG. 3.

représente 1'ADN du cosmide pHC 79 dans lequel on a fait apparaître le site cos.In this diagram, the svw part represents the cos site of a phage A; the meanings of the other elements of the diagram being identical to what has been indicated for FIG. 1, that is to say that the part

represents the DNA of the cosmid pHC 79 in which the cos site has been shown.

 The plasmid pHCG 3 is used to introduce the nif genes into a yeast by operating as follows.

Example 4
Preparation of the cosmid pPC 857
A - Source of nif DNA
The source of nif DNA is the plasmid pCE 1 (Km Tc Cb Gnd His Nif Tra IncP) described in ELMERICH C.,
HOUMARD J., SIBOLD L., RNHEIMER I. and CUARPIN N.,
Molec. Gen. Broom. 165, 181-189, 1978, which carries the nif genes of K. pneumoniae. These genes, 13 of which have so far been identified (MERRICK M., FILSER M.,
DIXON R., ELMERICH C., SIBOLD L. and HOUMARD J.,
J. Gener. Microbiol. 117, 509-520, 1980), are carried by two contiguous Hind III restriction fragments (PUHLER A., BURKARDT HJ and KLIPP W., Molec. Gen.

Broom. 176, 17-24, 1979). The plasmid pCE 1, introduced into E. coli SB 1801 (A (his gnd) rpsl) was purified by the method of HUMPHREYS GO, WILLSHAW GA,
ANDERSON ES, Biochim. Biophys. Acta, 383, 457-463, 1975. From 15 liters of culture in LB medium (Luria
Broth) I 370C was obtained 500 ug of purified DNA. A 5 g aliquot was hydrolyzed by the Hind III endonuclease (BIOLABS, 1 unit) for 30 minutes at 370C in the buffer recommended by BIOLABS.

B - Preparation of the vector
The vector chosen is the cosmid pHC 79 mentioned above. The vector DNA was purified as described in A from 200 ml of a culture in LB medium at 370C of the strain 5 K of E. coli, described in Proc. Nat. Acad. Sc. USA, 1978, 75, 4242, containing pHC 79 described in the article by COLLINS J. (Methods in Enzymol., 1979, 68, 309) after amplification with chloramphenicol (150 pg / ml). 200 µg of purified DNA were obtained. An aliquot part (1 μg) was hydrolyzed by the Hind III endonuclease as described in A.

C - Cloning
2 μg of hydrolyzed DNA of pCE 1 obtained in A and 0.5 μg of hydrolyzed DNA of pHC 79 obtained in B are mixed in 20 μl of ligation buffer (MILES L BORATORIES) to which 0.1 μl are added. 'a T4 ligase solution (MILES LABORATORIES). -The solution is incubated for 16 hours at 120C.

 The recombinant DNA is packaged in vitro in bacteriophage X according to the method of COLLINS J.

and HOHN B. Proc. Natl. Acad. Sci. 75, 4242-4246, 1978.

The phages thus prepared serve to infect strain C 600 of E. coli. The recombinant clones are selected for their character of resistance to ampicillin on solid LB medium containing 100 μg / ml of ampicillin.

 On 500 clones examined, the presence of nif genes was detected in 10-20% of the clones by hybridization with the DNA of small amplifiable plasmids carrying certain nif genes such as pSA 30 and pCM 1 mentioned in CANNON F., RIEDEL G.E.

and AUSUBEL F.M., Molec. Gen. Broom. 174, 59-66, 1979.

 Twenty cosmids from clones giving a positive hybridization reaction were purified and their restriction diagrams, after hydrolysis with Hind III and Eco R1, were established.

 The cosmid pPC 857 is represented in FIG. 4. It has been deposited at the CNCM under the number I-131; it was found to contain all of the nif genes.

 In this diagram, the Eco R1 sites are represented by: | and the Hind III sites are represented by: t.

 The nif genes are indicated by their common abbreviations: BALFGVSUNEKDHJ.

These genes are carried by two contiguous Hind III restriction fragments, the restriction site
Hind III separation being located between genes
K and D. In this cosmid pPC 857, the two Hind III restriction fragments carrying all of the nif genes are present but ligated in the wrong direction.

un site de restriction Eco R1. Les autres sites de restriction portés sur 1'ADN codant pour les gènes nif ne sont pas représentés sur le schéma.The cosmid DNA of pHC 79 is represented diagrammatically between two Hind III restriction sites. The cosmid DNA of pHC 79 contains, in addition to the cos site

an Eco R1 restriction site. The other restriction sites carried on the DNA coding for the nif genes are not shown in the diagram.

Example 5
Preparation of the cosmids pPC 874 and pPC 922
Starting from pPC 857 DNA, the operations of hydrolysis, ligation, packaging and selection of clones described in Example 4 were repeated.

Out of 200 clones examined, 9 were found to be nitrogen-fixing. The structure of the cosmids contained in these clones was established on the basis of their restriction diagram after hydrolysis with Hind III, Eco R1,
Bam H1, Bgl II (BIOLABS). They fall into two possible theoretical groups
one corresponds to I pPC 874 (shown in FIG. 5) which does not confer resistance to tetracycline,
- the other corresponds to I pPC 922 (shown in FIG. 6) which confers low resistance to tetracycline.

 In order to study the expression of the nif genes carried by these plasmids, they were transferred into bacterial strains of E. coli and K. pneumoniae lacking nif genes. We used, for example, the strain K. pneumoniae UNF 107 which carries a total deletion of the nif genes (Anif) described in Molec. Gen.

Genet., 1978, 165, 181.

 To transfer the plasmids pPC 874 and pPC 922 to K. pneumoniae, a lytic infection of the strains of E is carried out. coli which carry them by the phage Sh 434 cts. The lysate obtained is used to infect the strains of K. pneumoniae and the clones carrying the cosmid are selected by their resistance to ampicillin.

The nitrogen-fixing activist, according to the method described in Molec. Gen. Genet., 1978, 165, 181-189 (acetylene reduction test) by E. coli or from
K. pneumoniae (Anif) carrying the plasmids pPC 874 or pPC 922 is equivalent to that of the wild strain M5 al of K. pneumoniae from which the cloned nif genes are derived.

 To avoid loss of plasmids during the growth of host bacteria (segregation), ampicillin must be added to all media.

Example 6
Preparation of the cosmid pGPC 875
A - Source of nif DNA From 23 μg of pPC 857 DNA hydrolyzed by Hind III (6 BIOLABS units), the two restriction segments carrying all of the nif genes were purified by sucrose gradient according to the method of MANIATIS T., HARDISON RC, LACY E.,
LAUER J., O'CONNELL C. and QUON D., Cell. 15, 687-701, 1978.

B - HydrolZse of the vector
1 µg of the plasmid pHCG 3 DNA, prepared as described in Example 3, was hydrolyzed with Hind III (1 BIOLABS unit).

C - Cloning
The same operations as those described in Example 4 (ligation, packaging, selection of clones) were carried out using a mixture containing 1 μg of nif DNA and 0.5 μg of pHCG 3 DNA.

Out of 180 clones examined, two had the property of fixing nitrogen. Both have the same structure which is shown in Figure 7. The corresponding cosmid is called pGPC 875.

In this FIG. 7, the solid line represents the Hind III restriction fragments of DNA from
K. pneumoniae carrying the nif genes indicated by their conventional abbreviations.

 The Hind III Eco R1 restriction fragment of pHC 79 (6.5 Xb) is shown in dotted lines, as for the yeast block, it is represented by a wavy line (nsxm).

 This yeast block comprises the DNA of the URA3 gene and the DNA fragment of the plasmid 2ter1 H3 of form B (described I in Example 1-B).

Example 7
Yeast transformation
1 pg of pGPC 875 DNA was used to transform
S. cerevisiae, strain FL 100, according to the method of
GERBAUD C., FOURNIER Ph. BLANC H., AIGLE M. HESLOT H.

and GUERINEAU M., Gene, 5, 233-253, 1979. About 250 transformants selected for the character Ura + were obtained. The circular DNA contained in one of these clones (clone 3) was extracted according to the method described in the article by GERBAUD mentioned previously and was used to transform the strain 1106 (met, hsdR, hsdM) of E. coli according to the method of
COHEN SN, CHANG ACY and HSU L., Proc. Natl. Acad
Sci. 69, 2110-2114, 1972. The transformants were selected for resistance to ampicillin.

Out of 10 clones examined, all were found to be nitrogen-fixing. The structure of the plasmids carried by these 10 clones is identical to that of the starting plasmid pGPC 875. Thanks to the use of phageXrh434 cts as in Example 5, these plasmids were transferred back to different strains of K. pneumoniae mutated or deleted in the nif genes and in the strain
DB 6656 (pyrF Mu LacZam, trPam, hsdR) from E. coli.

It was thus possible to verify that the phenotypes of the cosmid pGPC 875 Ura + and nif had been preserved.

Nitrogen fixation
The nitrogen-fixing activity of E. coli or
K. pneumoniae (d nif) carrying the plasmid pGPC 875 is equivalent to that of the wild strain M5al of
K. pneumoniae.

 The plasmid pGPC 875 according to the present invention can therefore be used to transform a microorganism, - bacteria or yeast, not containing the genes responsible for nitrogen fixation.

 The plasmid pG 63 was deposited under the nO I-130 in the National Collection of Cultures of Microorganisms (CNCM) on August 25, 1980.

 The cosmid pPC 857 was deposited under No. I-131 in the National Collection of Cultures of Microorganisms on August 25, 1980.

Claims (11)

 1) Hybrid plasmid characterized in that it comprises at least - a DNA fragment of a 2 μm yeast plasmid, - a DNA segment containing a yeast gene, - a DNA fragment of a plasmid bacterial, and - a DNA fragment containing the genes responsible  of nitrogen fixation.  2) Hybrid plasmid according to claim 1, characterized in that the DNA fragment of the bacterial plasmid carries one or more cos sites of phage A.  3) Hybrid plasmid according to one of claims 1 and 2, characterized in that the DNA fragment containing all of the genes responsible for nitrogen fixation consists of two Hind III restriction fragments.  4) Hybrid plasmid according to claim 3, characterized in that the DNA fragment containing all of the genes responsible for nitrogen fixation comes from K. pneumoniae.  5) Hybrid plasmid according to one of claims 1 I 4, characterized in that the DNA fragment of plasmid 2 pm is the restriction fragment R1 H3 of 2.12 Kb of form B of the plasmid 2 pm.  6) Hybrid plasmid according to one of claims 2 I 5, characterized in that the DNA fragment of a bacterial plasmid is the restriction fragment Eco R1 Hind III of cosmid pHC 79 comprising the cos site.  7) Hybrid plasmid according to claim 1, characterized in that the yeast gene of the DNA segment is the URA3 + gene.  8) Hybrid plasmid according to one of claims 1 to 7, characterized in that the yeast DNA fragments are contiguous.  9) Cells and microorganisms comprising a hybrid plasmid according to one of claims 1 8.  10) A method of constructing the hybrid plasmid according to claim l, characterized in that it consists of using the plasmid pHCG 3, I adding purified nif DNA from pPC 857, I ligating, packaging in vitro in a phage X, and I infect from the hybrid plasmid thus obtained.  11) Process for preparing strains of yeast initially A nif transformed into nif +, characterized in that DNA extracted from bacterial clones (E. coli) having been infected with the hybrid plasmid prepared by the method according to claim 10.