DE3222142A1 - Process for the preparation of obligately methylotrophic bacteria, and plasmids and host organisms suitable therefor - Google Patents

Abstract

A hybrid plasmid with the replicon intrinsic to the methylotrophic bacterium is obtained from a plasmid from obligately methylotrophic bacteria, e.g. Methylomonas, on the one hand and a plasmid with selection markers on the other hand. This hybrid plasmid is - where appropriate after previous insertion of further genetic information - introduced by transformation into a host organism such as E. coli. After amplification and appropriate selection, the clones are treated with a suitable conjugative plasmid, and the mobilisability defect is eliminated by introducing another suitable plasmid. The clones obtained in this way are conjugated with plasmid-free obligately methylotrophic bateria as recipient, resulting - after selection - in clones which express proteins in accordance with the inserted genetic information.

Description

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HOECHST AKTIENGESELLSCHAFT HOE 82 / F 127 Dr.KL / mü

Process for the production of obligate methylotrophic bacteria and suitable plasmids and host organisms

The invention relates to the genetic manipulation of methylotrophic microorganisms, namely a method for Production of obligate methylotrophic bacteria that express the foreign DNA it contains. The inventive The method is characterized in that a) from an obligate methylotrophic bacterium, a plasma

mid isolated,
b) a hybrid plasmid with a replicon of the obligate methylotrophic bacterium produced therefrom and from a plasmid with selection marker core, c) this hybrid plasmid by transformation into a

Introduces host organism and amplifies it there, d) after selection the clones are treated with a suitable conjugative plasmid and the mobilization defect is eliminated,
e) the clones obtained in this way with pla3tnid-free obligate

methylotrophic bacteria conjugated as recipient and f) the desired clones selected.

The plasmid used in process step a) is preferably from a bacterium of the genus Methylomonas, in particular of the species Methylomonas clara isolated. Particularly the plasmid pBE 3 from the Methylomonas clara strain, which is available from the German Collection of Microorganisms, is preferred is deposited under the number DSM 2397. 25th

The plasmid used in process step b) with selection markers can be the plasmid pBR 322, which is described in Gene 2 (1977) 95-113. The so obtained Hybrid plasmids are referred to below as pRM.

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As a plasmid with selection niches, which in process step b) can be used, a hybrid plasmid is also suitable, which contains the genetic information for the expression of insulin, as described in the European patent application 0 032 675 is described.

The hybrid plasmid obtained after process step b) is then introduced by transformation into a suitable host organism, advantageously Escherichia coli, and there araplified.

In process step d), a suitable conjugative plasma, advantageously RP H, is introduced into this host organism which contains the hybrid plasmid. In addition, the mobilization defect is complemented by introducing suitable plasmids such as Col K and CoI y.

The host organism prepared in this way can then be used in process step e) the hybrid plasmid introduced in process step c) by conjugation onto a plasmid-free one obligate methylotrophic bacterium transferred as recipient. Preferred recipients are bacteria of the genus Methylornonas, in particular of the species Methylomonas clara, mainly from the ATCC 31 226 strain.

Finally, in process step f), the desired clones are cultivated in a medium that contains methanol as carbon source, as well as selected on the basis of the resistance or sensitivity to antibiotics transferred. The so obtained obligate inethylotrophen bacteria are able to the contained Frernd-DNA cxprirnieren, so for example to produce insulin.

The invention also relates to the plasmid from the Methylornonas clara-Starcm DSM 2397 and the hybrid plasraids with an obligate methylotrophic bacterium of its own

Replicon, i.e. plasraide, found in the bacteria of the genus Methylomonas, preferably of the species Methylomonas clara, in particular the strain DSM 2397 are replicated in which prokaryotic or eukaryotic DNA is integrated, in particular the one for the expression of insulin.

The invention also relates to the host organisms which contain the plasmids mentioned, as well as the host organisms, which also use the conjugative plasmid and those which also use plasraids to abolish the Mobilization defect included. Preferred host organisms contain RP 4 as conjugative plasmid and as plasmids to eliminate the mobilization defect Col K or Col V.

The advantage of the invention is that from a replicon of a plasmid from an obligate methylotrophic bacterium, so with a very narrow host area, use is made. The method according to the invention is thus distinguished characterized by high security so that it is applicable to recombinant DNA containing high-risk information.

The invention is explained in more detail in the following examples. Percentages relate to the weight, unless otherwise stated. . 25th

The following abbreviations were used in the examples:

ATP = adenosine triphosphate

EDTA = ethylenediamine tetraacetic acid or acetate (Ua) OD, -,.,. r Optical density at 600 nm TE buffer = aqueous solution containing 10 in moles per liter Tris-HCl, adjusted to pH 8, and 1 m mol of EDTA, also adjusted to pH 8.0 Tris (-HC1) = Tris-hydroxymethyl-aminomethane (-hydrochloride)

Examples "
Plasmid isolation from M. clara DSM

The plasmid isolation was carried out essentially according to the method of Humphreys et al. (BBA 383 (1975) ^ 57-463). For this purpose, the bacteria were centrifuged from 1 l of culture medium at an ODßnn of about 1.0 and the bacterial precipitate was resuspended in 5 ml of sucrose solution (25 % sucrose in 50 mmol / l Tris-HCl solution of pH 8.0). While cooling with ice, 1 ml of lysozyme solution (5 mg / ml lysozyme in 250 inMol / 1 Tris-HCl solution, pH 8.0) and 2 nil 0.2 molar EDTA solution of pH 8.0 were added. The mixture was incubated on ice for 5 minutes with occasional swirling. The lysis was then carried out by adding 8 ml of a mixture consisting of 50 mmoles / 1 Tris-HCl, 75.5 mmoles / 1 EDTA and 0.2 % of a nonionic surfactant ( ^v 'Triton X-100) with a pH of 8.0 , brought about. The highly viscous mixture was centrifuged at US 000 g for 30 minutes, a clear lysate being obtained as the supernatant. 1.1 ml of 5-molar saline solution and 1.1 g of polyethylene glycol with an average molecular weight of 6,000 were added per 10 ml of supernatant. The mixture was incubated at H ⁰ C overnight. The flaky precipitate was collected by centrifugation at 1,500 g for 5 minutes and dissolved in 3.5 ml of buffer solution (50 mmol / 1 Tris-HCl, 5 mmol / 1 EDTA, 50 mmol / 1 NaCl, pH 8.0) and that Volume of the solution measured. After adding 1 g of CsCl per ml of solution and 1/15 ml of a 1 Jigcn aqueous thidium bromide solution, the mixture was centrifuged at 16,000 g for 10 minutes. The supernatant from this centrifugation was centrifuged to equilibrium (20 hours at 11,000 revolutions per minute, 18 ° C., in a vertical rotor) and, even without irradiation with UV light, had two clearly visible bands about one centimeter apart r.uf. The fluorescent bands were emphasized by the irradiation of UV light with a wavelength of 365 nm

the lower plasmid band by side piercing of the Gradients are harvested with a needle and then the band is drawn up in a syringe. The ethidium bromide was removed by repeated shaking with isopropanol saturated with CsCl. After 3 times dialysis against 2 1 The plasmid DNA was added to TE buffer for at least 2 hours each obtained in a purity that allowed use in the following process steps.

If there were still impurities in the DNA solution, so this was twice with equal volumes of with 0.1 molar Tris-HCl solution of pH 8.0 and saturated phenol then extracted 3 times with absolute ether. The excess ether was blown off, the solution was 1/9 of the volume three molar sodium acetate solution added and the DNA through Addition of the same volume of isopropanol precipitated. After standing overnight, the mixture was on for 30 minutes Centrifuged 12,000 g and washed the DNA precipitate with 90% ethanol. The DNA was then lyophilized and recorded with TE buffer.

DNA isolation from aga rose

In the following procedure, the method used by Langridge et al. (Analytical Biochemistry 103 (1980) 264-271) Method application.

The DNA was applied to a horizontal 0.5 % \ &(> s Ap; aro3c gel (low-melting agarose, type VII, No. A- ^ 018, Sigma). After electrophoresis, the bands were visible by ethidium bromide staining made and excised from the gel. the Agarosesehei'ochen containing the DNA was melted at 70 ⁰ C in a glass tube, the volume measured, and cooled to 37 ° C. for this purpose, an equal volume amounts of the butanol and water phases described below were added.

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-β-

150 ml of n-butanol were shaken with 150 ml of v / water in the separating funnel and, after the phases had been separated, 1 g of hexadecyl-trimethylammonium bromide was dissolved in TOO ml of the water-saturated butanol phase. This solution was extracted with 100 ml dev water phase, a defoamer can be added (50 ul Antifoam A, Sigma). After the phases had separated overnight, they were collected separately.

The mixture obtained after the addition of these butanol and water phases was made by carefully turning the tube mixed thoroughly and waited for the phases to separate at 37 ° C. The upper butanol phase containing the DNA was separated off and the remaining aqueous phase was extracted twice in the manner described with butanol.

The combined butanol phases were 1/4 of the volume added 0.2 molar NaCi solution and mixed thoroughly again. The aqueous phase was separated off another salt extraction and the combined aqueous phases were added dropwise in the same volume Added chloroform (which had been purified on an alumina column). After standing on ice for half an hour the lower chloroform phase was discarded and remaining chloroform was blown out with air. The DMA was made with isopropanol precipitated and resuspended in TE buffer after separation.

Electrophoresis on polyacrylamide gel

8-15 polyacrylamide (PPA) gels were found to visualize and characterize DNA fragments below 0.3 MD Use. The gels were 1 mm thick, 30 cm long and 14 cm wide. The buffer used was a mixture which contained 89 mmoles of boric acid, 89 mmoles of Tris-HCl and 2.5 mmoles of EDTA per liter and had a pK 8.2.

To produce an 8% PAA gel, 33 ml of a 2 % PAA stock solution (23.22 g of acrylamide and 0.78 g of MjN'-methylenebisacrylamide in 100 g of aqueous solution), 10 ml

-f-

of the electrophoresis buffer mentioned, which, however, contained all components in a 10-fold concentration, 6.25 ml of a 6, H iigen 3'-ethylamino-propionitrile solution and 50.75 ml of water were added. The mixture was degassed at the water jet pump and the polymerization was started with solid ammonium peroxodisulfate. Immediately thereafter, the gel was poured and the electrophoresis was waited for at least 2 hours. Before applying the samples, a voltage of 100 V was applied to the gel for about one hour to remove residual ammonium peroxodisulfate.

The electrophoreses were carried out at 100 V and a current flow of 12 mA.
15th

Characterization of the plasmid pBE 3 from the Methylononas clara strain DSM 2397

The plasmid pBE 3 was digested with the restriction endonulcleases listed in Table 1 and the resulting Fragments separated by gel electrophoresis. By double digestions and by characterizing individual fragments cloned into pBR 322 - as described below the results set forth in Table 2 and Figure 1 were obtained.

a β »-a ·

-X-

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Table 1:

Overview of the enzymes used to characterize pBE 3:

enzyme

Number of fragments

Acc I. HI 1 Xo r II II 1 Eco RI EIl 5 Hinc II RV 5 Ava I. III 12th BaI I at least τ
α. 19th No division τ with Barn I. BgI I. Bs t II Eco I. Hind I. Hpa 1 Kpn I. No. u II Pst I. Pvu I. SaI I. Srna I. Sph Sst Ss t Stu Xba Xho Xran

XT-

Table 2:

Size of the fragments (in MD) produced by cleavage of pBE 3 with restriction nucM eases were obtained:

EcoR I : 4 I. , 0 Hinc II: : 7.2 : 2.43 Ava I: F1 : 3 : 1, , 09 H1 ■ : 1.42 : 1.94 Λ1 : 2.0 F2 : 1 : 1, , 27 H2 : 0.87 : 1.42 A2 • 1.72 F3 : 0 : 1, , 91 H3 : 0.26 : 1.27 A3 : 1, 26 F4 : 0 : 1, , 63 H4 : 0.18 : 0.91 A4a 1.03 F5 : O ₁ H5 : 0.63 A4 : 1.03 : 0, • 0.46 A5 • 0.85 : 0, 0.40 A6 • 0.66 : 0, 0.26 A7 0.55 • o, 0.18 A8 0.50 0, A9 0.13 0, A10 0.08 EcoR I 0, + EcoR I + Al 1. 0.07 Ava 0, •
• Hinc II: D1 0, 35 E1 D2 0, 22nd E2 D3a 0, 03 E3 D3 0, 03 E4 D4 85 E5 D5 66 E6 D6 66 E7 D7 63 E8 D8 55 E9 " D9 54 E10 D10 50 D11 40 D12: 14th D13. 13th D14: 08 D15: 07 D16: 05

- ICr -

Restriction Digests

Single digests were performed in a total volume of 50 to 100 μl using the manufacturers recommended buffers carried out. To ensure complete digestion of the DM, the samples were incubated overnight.

In the case of double and multiple digestions, the corresponding enzymes were usually added to the DNA at the same time. The buffer in these cases consisted of a solution containing 50 nmoles of sodium chloride, 5 mmoles of Tris-HCl (adjusted to pH 7.5), 6 millimoles of magnesium chloride, 6 millimoles of 2-mercaptoethanol and 100 mg Contained bovine serum albumin. Control experiments showed that the enzymes in this buffer give the same results as in those recommended by manufacturers. Exceptions were enzymes that had an above-average level Need salt. In these cases, digestion was first carried out with one enzyme at a low salt concentration and the second enzyme was only added after increasing the salt concentration.

Partial digestions with Eco RI

2. µg of plasmid DNA was incubated with 1 U of the restriction enzyme in a total volume of 50 µl at 37 ° C. Replicates were incubated different lengths and the reaction was stopped at various times by ten minutes of heating the mixture to 70 ⁰ C. A gel electrophoretic analysis of the respective band pattern showed how far the digestion had progressed after the defined times. :

Growing conditions

All Escherichia coli strains used were in L-Broth (10 g Bactro tryptone, 5 g yeast extract and 5 g table salt per

Liters of water). The Methylomonas clara starch were bred in one of the following two minimal media:

M 36: 1.5 methanol
0.1 % H ₃ PO ₄
0.083 % K ₂ SO ₁₄
0.018 % Na ₂ SO ₄ 10 H ₂ O 0.036 % MgSO ₁₁ 7 H ₂ O 0.00.4 % CaCO ₃

0.015 % citric acid

0.005 % (NH ₁ P ₂ Fe (SO ^) ₂ χ 6 H ₂ O

80, ul / 1 trace element solution

V 135 L: 1.5 % methanol

0.16 % K ₂ SO ₄

0.06 % MgSO ₁ , χ 7 H ₂ O 0.025 % Na ₂ SO ₄
0.014 ί CaCO ^

0.01 % Fe ₂ (SO ₁ P ₃

0.2 % H ₃ PO _n

0.28 5S NH ₃ (25 %)

0.3 ί ₃
0.3 % NaHCO ₃

1 ml / 1 trace element solution

Trace oil solution: 0.05 ζ / 1 H ₃ BO ₃ 0.01 g / l FiJ

0.0U g / 1 MnSO ₄ χ k 0.0H g / l ZnSO ₄ χ 7 0.02 g / l (NH ₁ P ₆ Mo ₇

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Construction of the hybrid vectors

The plasmid pBE 3 was with the restriction enzyme Eco R 1 partially digested so that the main part of the plasmid only has been cut once and in the linearized form Template. pBR 322 DNA was totally digested n; it Eco R I and subsequently subjected to treatment with alkaline phosphatase. The DNAs prepared in this way were added and incubated with T ^ DNA ligase at 14 ° C. overnight. 50, ug this ligated mixture was then used for transformation into the cells competently determined by CaCl2 treatment E. coli strain HB 101.

The bacteria were selected on L broth plates containing 20 _/ ug / ml tetracycline streaked and incubated overnight. Resistant colonies were inoculated onto fresh plates and well-grown clones were examined for the presence of Plasraid DNA with a higher molecular weight than pBR 322 by "single colony lysis". Clones containing such a plasmid DNA were finally cultured in 100 ml of L-broth with or without Tetracj ^r clin, and the plasmid DMA was obtained after the bacteria were stimulated with chlorampheniol.

Restriction digestions with the enzymes Eco R I and Ava I showed that the hybrid plasmids pRM 21 and pRM 54 shown in FIG. 2 had been obtained.

L ir: as o-ligf 'kt ion

Lignse reactions were carried out in 50 .mu.l volumes with a total DüA Korii .i.ntration of 20 μg _/ ml. Dev buffer contained 3 mmol Tris-HCl per liter (adjusted to pH 7.5), U ;: iMol magnesium chloride, 10 mMol dithioerythritol and 0.2 nmol ΛΤ ?. To diüson countenances was 1 _/ ul T ^ ~ ligase according

^l \ 00 U added; (1 U here corresponds to the amount of enzyme. The necessary; i ;; l to j0% of a Hind III-digested la: r.bda-DN'A in

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Ligate for 30 minutes at -16 ° C in a volume of 20 μl. The concentration of the DNA in this approach is about 330 μg / ml). The optimal ratio of the two DNAs to one another for a certain Liöaso experiment was calculated according to the method of Dugaiezyk et al., JMB 9_6 (1975) 171. Incubation was carried out at 1 H C for at least 16 hours.

conjugation

Recipient and donor were grown overnight to a ₀₀ ODG 1.0 to 1.2 (recipient) or 1, ¹ I - 1.6 (donor) grown and equal volumes of donor and recipient so placed together, that the mixture a large surface was available. The mixture was incubated for 2 hours at 37 ° C. without shaking and then 200 μl of it was spread on agar plates, the composition of which allowed selection against the donor and for the receptor provided with a new property mediated by the plasmid to be transferred. In the conjugation between E. coli KB 101 and M. clara DSM 2397, these were methanol miniature almediurn plates with the addition of an antibiotic (determined by the type of plaraid to be transferred).

In other experiments, recipient and donor were incubated as described above and 200 .mu.l of this mixture was streaked onto methanol-mini-calibrated-medium plates without antibiotics. These plates were incubated overnight at 37 ° C and the resulting lawn of bacteria with 2 ml of methanol Minimilmedium washed away. 200 μl of this suspension were then again smeared onto kethanol minimal medium plates: with the appropriate antibiotic. Clones appeared to ^l \ S 72stündigern incubating the plates at 37 ° C.

Expr ssion e eukary οtischer DNA in M. cla ra

The plasrnid pBE 3 was prepared in accordance with the procedures described in Derivatives of pBR 322 cloned or umklonierl, which as

copy

• ti

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c-D.N'A sequence of a eukaryotic gene that of monkey insulin contained.

Monkey insulin c-DNA is in the Pst! Site of pBR 322 derai'-t has been incorporated that this extraneous information under the control of the ß-lactamase pronotor in E. coli was expressed. This created a fusion protein that could be detected with anti-insulin antibodies (European patent application 0 032 675).

The monkey insulin c-DNA contains two internal Pst I interfaces. The coding information can therefore not be removed in intact form from the plasmid by Pst I digestion. In order to enable replication of this plasmid in M. clara, the procedure was therefore to incorporate DNA sequences from the M. clara plasmid pBE 3 into this insulin information-expressing pBR 322 derivative. Experiments were carried out as described above. In this case could be a series of hybrid Plasraiden be obtained, which differ in structure only in the pBR 322 portion built-monkey insulin C-DNA of the above-described hybrid vectors. The arrangement of the insulin gene in the pBR 322 part of these vectors remains unchanged in phase, so that these clones also express insulin-antigenic determinants in E. coli. One of these clones, which contains the entire pBE 3 sequence, was given the designation pINMc 68.

He; ? is known that the Mobilislerbarkeitsdnfekt of pBR by Plasrnide v / ie CoI C and V Col Komple / can ueröen nentiert (Young and Poulis, Gene H (1978) 175-179). Therefore, the plasmid Col K was also introduced by transformation into the above-described donor strains made competent by calcium chloride treatment with the conjugative plas.xid RP H and the hybrid vectors of the pRM series. As an indicator staran for the detection of the plasinide Col K , the

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Colicin-sensitive strain AB 1157 was used (Warren et al., MGG 170 (1979) 103-107). In order to select for clones in conjugation experiments between the E. coli donor strains HB 101 (RP ¹ J, Col K, pRM 5H) or HB 101 (RP 4, Col K, pRM 21) and the recipient M. clara ATCC 31226, which contained the hybrid vector was selected in the presence of high doses of tetracycline (50 μg / ml on minimal methanol medium (M 36).

An analysis of numerous M. clara clones showed that in approx. 10 % of the cases clones had been obtained which contained only the hybrid vector. In a conjugation experiment between the E. coli donor strain HB 101 (RP H, Col K, plnMc 68) and the M. clara recipient ATCC 31226, M. clara clones containing only the plasmid PInMc 68 could be obtained in the same way .

The M. clara clones obtained with the plnMc 68 plasmid were subsequently bred and used via a radioiir.munassay or a fat cell assay checked for their insulin content. Corresponding to that in European patent application 0 032 Observations made with E. coli could also in the case of M. clara ATCC 31226 insulin values between 1 and 5 IU can be measured per liter. Thus, the insulin information contained in the pBR 322 portion of the hybrid vector plnMc 68 is also obtained also correctly and efficiently expressed in H. clara.

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Claims (22)

~ Ζ * 5 ~ H0E 82 / F 127 patent claims 1. A process for the production of obligate methylotrophic bacteria which express the foreign DNA contained therein, thereby marked that one a) a plasmid from an obligate methylotrophic bacterium isolated, b) from this and from a plasmid with selection marker core a hybrid plasmid with one of the obligate methylotrophs Bakteriura produces its own Replicon, c) introduces this hybrid plasmid into a host organism by transformation and amplifies it there, d) after selection the clones with a suitable conjugative Treated plasmid and corrected the mobilization defect, e) the clones thus obtained with plasmid-free obligate methylotrophic bacteria as recipient conjugated and f) the desired clones selected. 2. The method according to claim 1, characterized in that the obligate methylotrophic bacterium is one of the genus Methylomonas. 3. The method according to claim 2, characterized in that the bacterium is of the type Methylomonas clara. ^ 'The method according to claim 1 to 3, characterized in that the plasmid-free bacterium is from strain ATCC 31,226. 5. The method according to claim 1 to 3, characterized in that the bacterium from which the plasmid is isolated from. Sta: n: a DSM 2397 is. 6. The method according to claim 1 to 5, characterized in that the plasmid is pBR 322 with selection markers. -Tt- HOE 82 / F 127 -Ζ. 7. The method according to claim 1 to 6, characterized in that that the plasma raid with selection markers contains the genetic information for the expression of insulin. 8. The method according to claim 1 to 7, characterized in that the host organism is Escherichia coli. 9. The method according to claim 1 to 8, characterized in that that the conjugative plasmid is RP 4. 10 10. The method according to claim 1 to 9, characterized in that the Mobilisierbarkeitsdefekt by introducing the Plasmids Col K or Col V is resolved. 11. Plasraid from DSM 2397. 12. Hybrid plasmids with one obligate methylotroph Bacterium's own replicon. 13. The plasmid of claim 12 which is replicated in a bacterium of the genus Methylomonas. 14. Plasmid according to claim 13> in a bacterium of the Species Methylomonas clara is replicated. 15. Plasraid according to claim 15, which is replicated in a bacterium from strain DSM 2397. 16. Plasmid according to claim 12 to 15 with integrated eukaryotic DNA. 17. Plasmid according to claim 16 with integrated DJiA for the Expression of insulin. 18. Plasmid according to claim 12 to 15 with an integrated prokaryotic DNA. a- tr a * λ ty - ^ ⁸ - HOE 82 / F 127 19. Host organism containing a plasmid according to claim 12 to 18. 20. Organism according to claim 19 _S which contains a conjugative plasmid. 21. Organism according to claim 20, which contains RP H as conjugative Plasraid. 22. The organism of claim 21, which contains the Col K or Col V plasmid.