CA1270216A - Processes for the preparation of obligate methylotrophic bacteria which express foreign dna, and plasmids and host organisms suitable for these - Google Patents

Abstract

Abstract of the disclosure:

A hybrid plasmid with the replicon inherent to the methylotrophic bacterium is obtained from a plasmid from obligate methylotrophic bacteria, for example Me-thylomonas, on the one hand and a plasmid with selec-tion markers on the other hand. This hybrid plasmid is introduced by transformation into a host organism such as E. coli, if appropriate after previous introduction of further genetic information. After amplification and appropriate selection, the clones are treated with a suitable conjugative plasmid, and the mobilizability de-fect is abolished by introduction of a further suitable plasmid. The clones thus obtained are conjugated with, preferably plasmid-free, obligate methylotrophic bacte-ria as recipient, clones being obtained, after selec-tion, which express proteins corresponding to the gene-tic information introduced. It is also possible to pre-pare spheroplasts from the bacterium serving as the re-cipient and to insert the hybrid plasmids into the for-mer by transformation.

Description

2 - HOE 8~/F 1~7 K

The invention relates to the ~ene~ic manipula- .
tion o~ me~hylotrophic microorganisms~ in par~icular pro~
cesses ror the preparcition of obligate methyLotrophic bac~eria which express contained foreign DNA, plasmids for introducing the ~oreign DNA, and hos~ or,~Janisms. One of ~he proce7,ses according ~o the inven~ion comprises a~ ;sola~in~ a plasrnid which orisinates from cin obligate me~hylotrophic ~arterium, b) preparing from ~his and ~rom a plasmid wi~h selection 10 markers a hybrid plasmid w;th a repl;con inheren~ ~o ~he obligate methylo~rophic ~acterium, c) introducing ~hls hybrid plasmid by transform2tion in~
~o a hos~ organism and ampl;fy;ng i~ ~here, d) a~ter selec~;on, trea~;ng the clones w;~h a su;table ~5 conjugat;ve plasm-id ancl abol;sh;ng the mob;li~ability defect of ~he hybr;o plasmld, e) conjuga~;ng the clones ~hus obtained wi~h obligate methylotrophic bac~er;a as the rec;p;erit and f) selec~ing ~he des;re~ clones r lhe plasmid ernployeci ;n process step a) is pre~
~er~abiy isola~ed ~rom a ~ac~erium of ~he~~enus '.~'e~hy~
omonas, in particular o~ ~he spec;es ~e~hylomonas clara.
The plasmic, p~E 3 from ~he M~ra~hylorilonas clara strain which is deposiLed in .~,e Germa,i Collec~ion of Microorgan- !
Z5 isms under the num~er DSM 23'~7 and appropria~e plasmids~
having the same repliçon are partiçularly preferred.

~7~3~
The pLasmid w;th selec~ion markers which is em^-ployed in process s-;ep b) can be the plasmid pBR 3~2 which is described in Gene 2 (1977) ~5-1130 The hybrid plasmids ~hus ob~ained are denoted pRMX in the tex~
5 which follows n A hybrid plasmid which contains the genetic infor--mat;on for ~he expression of insulin, as is dPscribed in European Pa~ent Application 0,032,675, is also suic~
able as a plasmid ~ith select;on markers whic17 can be employ2d in process scep b)~
The hybrid plasmid ob~tained by process step b) is then introduced into a suitable host or(~anism, advan~ta~eously Escherichia coli, by ~transforma-~;on, and ampl;fied there.
In process step d) a su;cable conju~ative plas~
m;d~ advanta~eously RP 4, ;s ;ntroduced ;nto this host organ;sm which conca;ns the hybr;d plasm;d. In addi t;on, .he mob;l;zabil;ty defect is complemer1ted by ;n~
troduct;on of sui1ta~le plasmids, such as Col K and Col V0 The host organism thus prepared can then~in pro~
cess step e), transfer by conjugation the hybrid plas-mid in~roduced in process s~ep c) ~to a,preferably plas-rr,;d~free, obligate metl1ylotrophic bacter;un as reci-pient~ Preferred rec;p;ents are barcer;a ~f ~he ~enus Me~hylomonas, in particular of the species Me~hylomonas clara, abcve all of the s~rain ATCC 31 ~26. This strain is described~ for example, in ~erman Patent 2,~33,451 and US Pa~en~ b,166,D~
- Firlally, in process s~ep f~ ~he desired clones ~ - 4 -are selected by culturing in z medium which contains methanol as the carbon source and on ~he basis of ~he transferred resistance or sensitivity ~owards antibio-~ics. The obliga~e methylotrophic bac.eria thus ob-tained are capable of expressing ~he con~ained foreign~NA, and thus, for example, of producing insulin.
De~ails of these process steps are expanded on uithin ~he framework of ~he examples.
Furthermore~ the invention relates to a varian~
of the above~,entioned process, in which the process steps d) and e) are simplified as follows:
After selection, in s~ep d), the hybrid plasmid is isola~
ted and, in step e), the isolated plasmid is introduced by ~ransformation into spheroplasts of the obligate me-thylotroph;c bacter;um.
Thus the ;nvent;on also relates to spheroplastsof methylotrophic bacteria, particularly of obligate me-thylotrophic bacter;a~ especially of bacteria of the ~enus Methylomor,as, preferably the species Methylomonas clara. The stra;n Methylomonas clara ATCC 3~2~6 is par~
ticularly preferred.
The spheroplasts according to ~he invention can be prepared by culturing the bac~eria in a glycine~ric~
medium con~aining an osmot;c stab;lizerO Th;s med;um is preferably sligh~ly hypo~on;cO Fur~her preferred em~
bodimen~-s of this preparation process are explained in more detail below.
Furthermore~ ~he ;nvention rela~es ~o the use of ~he ne~ spheroplas~s for inser~iny ex~ternal DN~ in-~o " ~7~L6 ~. 5 ~
these bac~eria. This external Dr~A is pri~aril~ in~ o-duced in the form o~ a plasmid. Suitable plasmids are the abovemen~ioned hybrid plasmids.
In order to produce ~he spheroplasts, ~he methy lotrophic bacteria are cultured in a sui~able medium, advantageously in a minimum me~ium, up to a suitable cell densi~yD Sui~able cell densities are in ~he OD600 range of r preferably, 0.5 to 1 ~, in par~icular 0.9 ~o 1.4. A defined volume of ~hese cul~ures is then in~ro-duced into about S ~imes the amoun~ of the m*dium~entloned, which is morec~ver ~lycine-rlch and conl:ains an osmot;c stabll;zer. The con~ent of glyc;ne can be as much as the saturat;on concentra~;on~ a content of 2 to 4~ by we;ght be;n~ pre~erred.
Su;~able osn~ot;c stab;lizers are sugars, such as sucrose, sugar base alcohols, such as sorb;tol, and polyglycols, such as polyethylene ~lycol 6000. The me-dium 1s preferably slightly hypotonic, this being brought about by suitable concentra~ions o~ the osm~ic s~ab;l;zer. ror example, a sucrose concen~ration of 10X
by weigh~ or 1 mol~r sorb;~ol is suitableO
~ he bacter;a are shaken in this medium un~il es-sen~;ally no rod~shaped me~hylo~rophic bac~eria are de-tec~able under the phase con~rast m;croscope~ In gene-ral, after about 1 hour of shaking (100 to 1gO rpm) atabou~ 37~, the produc~ion of the spheroplas~s as im-mobile spherical structures can be observedO rhis pro-cess is generally comple~e 3f~er abouc 4 hours~

The produred spheroplas~s are carefully ~7~
-- 6 ~
cen~rifuged c'own, for example at 2600 x g and 4C fcr 10 minu~es, and resuspendeci in su;table med;a. rhis re-suspension mediurn likew;se contains an osmo~ic stabili-zer at 3 sulta~le concentration, for example 15% by S weigh~ of polye~hylene glycol of rnolecular ~eight 6000, to stabilize the spheroplas~s.
A mixture which comprises equal par~s Qf the rnedium used to resuspel-d the spheroplasts and the car~
rier for the DNA to be introduced~ in a sui~able buf,err 1D is added to this suspension at an approximate volu~e ratio of 1:5 D A suitable DNA carrier is, as detailed above, primarily a hybrid plasm;d~ A buffer suitable for this purpose comprises an aqueous solu~ion containing 10 mmol of TRIS (tr;s~hydroxymethyl)aminomethane) and 1 mmol of sodium e~hylenediaminetetraacetate ~TE bu~
fer) per liter. An osmotic stabilizer is added to ~his mixture tfor example 4~ by weight of polyethylene glycol 6000 solut;onr three t;mes the volume oF the suspenslon of spheroplasts) ana it ;s carefully and ~horoughly mixed. After standing briefly at room te~pera~ure~ the resuspension medium, about ten times the volume of ~he suspension of spheroplas~s, is added and the mixture i5 carefully centrifuged ~3600 x 9).
1he precipitate is then taken up in resuspension med;urn, twice the volume of the suspension of sphero-plas~s, and s~reaked on~o agar plates to check ~he~her transfor~a~ion nas ~aken place. These aaar pla.es have a composition correspond;ng to the resuspens1On me~iurn and con~ain, in addi~ion to 1~5~ by weiyh~ of Bac~o agar, an arlt;ob;o~;c suit~ble for selec~ion, for example 5C IAg /rnl of ampicillin or 10 ~g/ml of tetracycl;ne, in tl-e case where ~he hybrid pla~ids employed contain~d the approp-r;ate resis~ance genes~ The plates are ~hen incubated a~ 37C for 1 to 3 days, and resis~ant colonies~ af~er being cultured in ~hç abovemen~ioned liquid medium wh1ch contains 2 suitable antibio~ic, are investi~ated for ~he presence of plasmid DNA (Humphreys e~ al~, BBA 383 (1975) 457-463). By th;s means, the plasmids ernployed 10 for transforma~;ion can be isola~ed from the me~hylotro-phic bac~eria.
~ ur~hermore, the invention relates ~o the plas-mids from the Mer.hylomonas clara strain DSM 2397 and the hybrid plasrnids with a replicon inherent to an obligate r,~thylo~rophic ba~ter;um, jM other words plas~i~s ~Ih,~
are repl;cated ;ll the bac~eria of the ~enus 'Methylomo-nas~ preFerably of the species ~lethylomonas clara, in part;cular the stra;n ~TCC 312~6, ;n ~h;ch prokaryotic or ~ukaryotic DN~ is in~egra~ed, in particular ~hat for the express;on of insulln~
Fur~hermore, ~he inven~ion rela~es ~o ~he host organisms which con~ain the pLasmids mentioned, and ~he hos~ organisms which addi~ionally con~ain the conju~a-~ive plasmid and~ furthermore, those which con~ain addi-~ional plasmids for the aboli~ion of the mobilizationdefectD Pre-ferred host organisms contain RP 4 as ~he conju~ative plasmid and Col K or Col V as plasmids for ~he a~olition of ~he mobili~a~iorl de^fec~
The acJvan~age of the invention comprises use ' 2~6 be;ng made of a replicon of ~ plas~id fro~n an obliQOte me~hylotroph;c bac~erium, in other words one ~i.h a very r~arrow ran~é of hosts Thus the process according to the invent1On is distinguished by a high degree o~ safe-ty, so that it can be applie~ ~o recorlbinant bN~ which contains hazardous information.
~ he invention is illustrated in more de~ail in the following examples. In these, percentage da~a re-late to weight unless otherw;se specified~
In th~ examples, the following abbreviations are used:
ATP = adenosine triphosphate EDTA ~ ~thylenediarn;netetraacetic acid or -acetate tNa) OD6~0 = optical density at 600 nm TE-buffer = aqueous solution containing 10 mmol of tris HCl per l;ter, adjusted ~o pH 8, and 1 mmol o~ EDTA per liter, also adjusted to pH 8.0 Tris ~HCl)- trishydroxymethylam;nomethane ~hydrochlo-r;~e).

In the Drawin~s Fiqure 1 shows a restriction chart of plasmid pBE 3 isolated from the strain Methylomonas clara DSM 2397; and .~ .
Figures 2 and 3 show hybrid plasrnids containing the complete DNA of plasmid pBE 3 and DNA from plasmid pBR 322, the latter being inserted in both of the possible directions.

- 8a Examples 1. Isolation of plasmid from M. clara DSM 2397 The plasmid was isolated essentially according to the method of Humphreys et al. (BBA 383 457-463).
Fox this purpose, the bacteria from 1 liter of culture medium having an OD600 of about 1.0 were centrifuged down and the precipitate of bacteria was resuspended in 5 ml of sucrose solution (25% sucrose in 50 mmol/l of tris HCl solution of pH 8.0). While cooling in ice, 1 ml of lysozyme solution (5 mg/ml of lysozyme in 250 76~
9 _ rnmol/l of ~ris I~Cl s~lu~ion~ p~ &~Oj and 2 ml o~
0~2 molar EDT~ solu~ion of p~l 8.0 ~ere ad~ed. ~he mix-~ure wa~ incuba~e~ on ice ~or 5 r~inutes with occasional swirlin~ Lysis was ~hen brough~ abou~ by the addi~ion S of 8 ml of a mix~ure con.ain;ng S0 mmol/l of cri H~l, 75O5 mrnol/L of EDT~ and 0.2% of 2 nOn-iOlliC
surFac~ar)t ((R)Tricon X~100) of pH ~Ø The highly viscous mixture was centrifuged at ~8~0D0 x ~ for 3~ rni~
nu~es, a clear lysa~e being obtained as the supernatant.
10 Per 1D rnl o-F supernatan~, 101 ml of 5 molar saline and 1~1 9 o~ polyethylene glycol, of meall molecular weight ~,Oi30, were added. Tlle m;xture was incubated overnight at 4~
The flocculent precipitate was collected by centr;fuga-tion at 1500 x ~ for 5 minutes and d;ssolved in 3.~ ml 1S o~ buFfer solu~cion tS0 mmol/l of tris HCl~ 5 mmol/l of NaCl, pH 8~0), and the volume of the solution was measured. Af~er addition of 1 9 of CsCl per rnl of solut;on and of 1/15 ml of a 1% strength aqueous ethi d;LIm bronl;de solution~ centrifugation uas carried out for 10 rninu~es at 16,0ûO x g. The supernatant frorn ~chis was cen~rifuged to equilibr;um (~0 hours at 47,090 .revo-lut,ons per rnîllu~e, 18C, in a Yertical rotor) and showed ~r~o bands which were clearly visible, even ~i~h_ ou~c irra~iation ~ith U~ ligh~c, lying abou~c one centime-~er apar~c. rhe ~luorescen~ bands were accentuated byirradiation with UV light of wavelength 366 nm~ and the lower pl.asmid band was harves~ed by piercing che gra-dien~ 1rorrl ~he side ~ith a needle and subsequen.ly aspi--ratin~l ~he barld ;n a syringe. rhe e~hid;um bromiàe was z~z~

removed by repeated extrac~ion by shaking with i50pr~pa~
nol sa~ura~ed wi~h CsCl. Af~er dialysis a0ainst 2 liters o~ TE buffer 3 tiloes~ for at leas~ ~ hours each time, ~he plasrnid DNA was ob~ained in a puri~y 5 ~h;ch permitted i~s use in the folluwing prccess steps.
I~ impurities were s~ill eviden~ in ~he DNA ~o-lucion, i~ was excracted twice with iden~ical volumes of phenol sa~ura~ed wi~h 0.1 rnolar ~ris HCl solu~ion of pl' 8.0 and then 3 ~imes with absolute e~her~ The excess e~her was blown off. 1/~ of the volume of ~hree rnolar sod;um acetate solution was added to ~he solution, and the DNA was precipi~ca~ed by add;~ion of the same volume of ;sopropanol~ After standing overnigh~, the mixture was centrifu~ed a~ 12~000 x ~ fùr 30 minu~es~ and the DNA precip;~ate was washed w1th ~0X s~rength ethanol.
Subseque~tly, the PNA ~as freeze~dr;ed and .a~en up ;n TE buf~er.
2. Isolation of DNA froln ac~arose In ~cl~e process which follows, the method descri~ed by Lan~ridge et al. (Analytical Biochemistry 103 ~1980) 264-271) was used.
The DNA was applied ~o a horizon~cal 0.5% strengtl agarose ~el (low~melting a~arose, ~ype VII, NoO A-~018, S;~rna). hfter elec~rophoresis, the bands were visualized by s~aininy wi~h e~hidiurll bromide and cu~ ou~ of ~he gel.
~he agarose disl con~aining ~he D~A was melted in a glass ~ube a~ 70C~ and the volume was measured, and it was cooled CIGwn ~o 37~C. To ~his were added es1ual volu~es -~ o~ the bu~anol and wa~er phases described below.

- ~.;27~
1 1 ^
150 ml of n~bucanol were shaken with 150 n~l of wa~er in a separating funnel,and~ af~er separ&tioR OT
the phases, 1 ~ of hexadecyltrinletilyLalllMon;um bromide was dissolved in 100 ml of the water~saturated butanol S phasea This solucion was ex~racted by shak1ns with 100 ml o~ the wacer phase, i~ being possible f~r a defoamer to be added (50 ~1 of An~ifoam A, Sigma). After separa~
tion of ~he phases overnight~ they were collected sepa-rately.
1~ The mix~ure obta1ned af~er addi~ion of these bu~
tanol and water phases was thoroughly mixed by carefully rotating the tube, and the phases were allowed to sepa-rate at 37C. The uppe~r butanol phase con~ain;ns ~he DNA was separa~ed off, and the remaining aqueous phase 1~ was extracted twice more w;~h bu~anol in the manner des~
cribe~ One quar~er of the volume of 0.2 molar NaCl solut;on was added to ~he combined butanol phases, and thorough m;xing was again carried out. Af~er ~he aqueous phase had been separa~ed o~f, another salt ex~raction was 2n carried out and ~he same volume of chloroform (which had been purified over an aluminu~ oxide column) ~as added dropwise to ~che combined aqueous phases~ After standin~
on ice for half an hour, the lower chloroform phase was discarded and ~he remain1ng chloroform ~!as blown ou~
wi~h air. The DNA was precipitated wi~h isopropanol and, af~er separat;ng off~ resuspended in TE buffer.

3. El~ p~ si_ o__E~ a _ yla~,ide~
8 ~ s~crength polyacrylamide (PhA~ ~3els ~Jere used for ~he visual;~acion and character;za.10n of DNA

2~i ~ 12 -fragmen~s below 093 MD. The sels were 1 mm thick, 3~ cr, long and 14 cm wide. A mix~ure contatning ~9 rnrnol oF
boric acid~ ~ mmol o^f tris HCl and 2.5 mrnol o~f EDTA
per L1~er and haviny a pH of 802~ served as ~he buffer.
S In order to prepare an 8~ PAA ~el, 33 ~l of a 24% s~rength PAA stock solu~ion t23.Z2 9 of acrylamide and 0.78 9 of ~ methylenebisacrylamide in 100 g of aqueous solut;on), 10 ml of the electrc)phoresis buffer men~ioned, ~hich however con~ained aLL componen~s in 1n 10-fold concentr2~ion~ 6.25 mL of a 6~/t% s~reng~h 3~
ethylaminopropioni~ri Le solution and 50.75 ml o~ wa~er ~ere mixed. The mixture was degased on a ~a~er pump, and polymerization was started wi~k solid ammonium peroxodi sulfate. The gel was poured immediately thereaf~er, and a m;nimum oF 2 hours was allowed to elapse before the start of elec~rophoresis. Before apply;ng ~he samples, a voltage of 100 V was applied for about one hour ~o the gel to rernove resi~ual ammonium peroxodisulFate.
The eLectrophoreses were carr;ed ou~ a~ 100 V
and a current of 12 mA.

4. C_aracterizat on of ~h~lasmid ~BE 3 from the in ~S~ 23~7 The plasmid pBE 3 was digested wi~h ~he re~
str;ction endonucleases men~ioned in Table 1, and the ~5 fragments produced were separated by geL electrophore \~ SiSn The results recorded in Table 2 ancl Figure ~ ~ere ob~ained by double diges~ions and by charac~erizat;on o~
;ndividual fragmen~s cloned in pE~ 322~ as described below.

%7~21~
~ 13 o - The plasmid pBE 3 is a dele~ion mu~c-7n~ o~ a larger plasmid~ Besides ~hisr smaller plasmids also exist. All ~hese plasrl7ids are equivalen~ in ~he sense oF ~his inven~ion when ~hey con~ain the replicon inhe~ ¦
S rent to Methylolnor7as.
rah le 1: ~
Survey of the enzymes used for ~he charac~eriza~ion o~ ¦
pBE 3:
Enzyme Num~r oF ~ragment~ I
10 Acc I
Xor II
Eco RI
H;nc II S
Ava I 12 15 ~al I a~ leas~

No cleav3ge with ~am HI
P9l II
~0 E3st EII
Eco RV
Hind IXI
l1pa I
~pn I
Nru I
Pst I
Pvu II
Sal I
Sma I

gLZ~Z~6 ~ 14 -- Table 1 ~continued) Sph I
Sst I
~st II
Stu I
Xba I
Xho I
X~n I

Table 2:
__ _~_ Size o~ the frag~ents ~;n MD) wh;ch were produced by cleavage of p~E 3 wî~h restriction nucleases:
EcoR I: H;nc XI- Ava X:
F1:4.14 H1 : 6.8 A1: 2.0 1 5 F2 :3 ~ 09 H2 : 1 . 42 A2 : 1 ~ 72 F3 :1 .27 H3 : 0 . 87 h3 : 1 .26 F4 ~1 H4 : 0.31 A4a: 1003 FS:0.63 H5 : 0.28 A4: 1.03 A5: 0.85 ~ A6 : 0 . 66 A7: 0.55 A8: 0.50 A9: 0.13 A1 0: 0~08 A11: 0 ~ 07 EcoR I~ EcoR I~
hva I: Hinc II:
D1 : 1 r35 E1 1 2 . Z9 D2 : 1, Z2 E2 : 1 ~ 83 - ~2~2~6 Table 2 (con~inued) D3a: 1.03 E3 : 1~42 D3 : 1.0-~ E4 : 1.Z7 D4 : 0O85 E5 : ~).91 D~ : 0.70 E6 : 0.63 D6 : 0.63 E7 : 0.~6 D7 . 0~55 E8 : O./tO
D8 : 0.5~ E9 : 0.31 b~ : ODS3 E10: 0O2 D10O 0~50 D11: 0~4 D12: 0.1~
D13: 0~12 D14: 0~1 : O.i D16: 0.1 S. Res~ric~ion diges~;ons 'Cnd;vidual diges~;ons were carr;ed ou~ in a ~o~
tal volume oF 50 to 100 ~l with ~he hufFers recommended by ~he manufac~urers. In order ~o ensure complete cii ' gestion o; the DNA, ~he samples were incu~ateci over~
n;gh~.
For double and multiple ciiges~ions~ ~he approp~
2.5 ria~e enzymes ~ere usually adcied ~oge~her ~o ~he DNA.
In these cases, ~hP buffer comprised a solution contain~
;ng 50 mmol o~ sodium chloride, 5 mmol of ~ris HCl (ad~us~ed to p~ 7.5~ 6 mmol of ~agnesh!m chLoride, 6 mmol of 2~mercap~oethânol and 100 rn~ of bovine serum 2~2~i ~ 1b albumin per liter. Control experirnents showed that ~he enzymes provioe the same resul~s in this buffer as in those recolnmended by the rnanufacturers. The exceptions were enzymes whi~h have an above average salt re~uire~
men~ In these cases, diges~ion was initially carriecl out at a low salt concen~ration wi~h one enzyme~ and the second enzyme was only added after increasing the salt conc E n~ration~
6~ Par~ic~l d;ges~ions wi~h Eco R I
2~ 9 of pLasmid DNA were in~ubated wi~h 1 U of the restric~ion enzyme a~ 37C in a ~o~al volume of 50 ~Al. Identical ~;xtures were incubated for differen~
times, and the reaction was s~opped at ~he various times hy heat;n~ the rnixture at 70C ~or ten minutes. Ana~
lysis of the par~icular pa~tern of bands by 0el electro phoresis showed the.extent to which ~he digest;on had advanced after the defined t;mes.
7. ~row~k ronditlons hll Escherichia coli s~ra;ns used were cultured in L broth ~10 9 of Bactro Trypton, 5 g o~ yeast e~rac~
and 5 Q of sodium chloride per 1 liter of water)~ The Me~hylomonas clara strains were cultured in one of the ~wo followlng minimum med-;a:
3~ 5% of me~hanol 0~1% of H~P04 0l0~3% o~ ~S04 O~D1~X of Na2S0~ H~0 0~3S% o~ ~S~4 O 7 ~l2 -0 004JO of CaC03 `" ~2702 G
~ ~, O.O~S~ of citric acid 0,0~5% of (NH~t)2Fe(S04)2 6 H20 ~o~ l of t,ace elemel1t solw~ion V 135 L D 1 L5% of methanol n.16~ of K2S04 ODO6% of MgS04 . 7 H20 tl.O2S~ Of Na2SO4 0.014% Of GaCO3 Q~01~ of F~2(S~4)3 ~0 0.2~ of ~13POf~
0.28% of NH3 (25%) 0.3YO of KN03 0.3X of NaHC03 . 1 mL/l of ~race element solution Trace element sol(~ion:
0.05 g/l of H~B0 0~01 g/l of KI
0~04 g~l of MnS04 . 4 t~20 0.~4 ~/l o~ Z~S04 . 7 ~
n.o2 g~l of (NH~)6Mo7024 . Construc~ion of ~he hybrld vectors The plasm;d pBE 3 was partially d19es~ed with the restriction enzyme Eco R I so tha~ ~he major part of the plasmid ~as only cut once and was presen~ in ~he linear form. pBR 322 DNA was comple~ely digested with Eco R I and ~hen subjected to treatmen~ with alkaline phosphatase. The DNAs thus prepared ~ere mixed and in~
cubated~wi~h T4 DN.~. ligase at 14C overnightD S'J~ of ~his liqa~ed mixture then served for transformation ;ntO

1~7~

~he E. coli s~rain ~l~ 101 made competen~ by treatmen~
w;th CaC l2 .
The bac~eria were smeared onto L broth pla~es with 20~/ml of tetracycline and incuba~ed overni~htr

5 Resistant colonies were reinoculated on fresh pla,es and well-gro~n clones were inves~igated for ~he presence of plasmid DNA having a higher molecul~r weight ~han p~R
322 by "single colony lysis"~ Clones !Jhich con~ained plasmid DNA of ~his type were ~inally cul~ured in 1Q0 ml of L broth wi~h or without tetracyclire~ and, after chLo~
ramphen;col stimula~ion of ~he bac~eria, ~he plasmi~ DNA
was o~tained.
Res.riction diges~ions with the enzymes Eco R I
and Ava I showed ~hat ~he hybrid plasrnid pRM 21 shown in Flgure 2 and the hybrid plasmid p~M ;4 sno~n ;n Fi-~ure 3 had been obtained.
In addition, it is poss;ble ~o identi~y hybrid plasm;~s containin~ Fractions of pBE 3.
9. Lic~as~ reac~ion _ _ __ _ L;gase reac~ions were carried ou~ in a volume of 50~ l at a total ~Nh concentrat;on of 20 ~g/rnl. 1he buffer contained 30 mmol o~ ~ris HCl (adjusted to pH
7~5), 4 mmol of ma~nesiurn chloride, lO mmol of dithio-ery~hri~ol and 0.2 mrnol of hrp per liter. 1~l o~ T~
2S li~ase~ correspondir,g 'LO 400 Ur was added to ~hese rnix tures tin this contex~, 1 U corresponds ~o the amount of enzyme necessary ~o liga~e 50% o~ lambda D~!A, irhich has been di~ested with H;nd III, ;n 30 minutes a~ ~16~C
in a volume o~ ~O~l. T~e concentra~ion of ~ e ~NA ir, ~7~z~

this mixture is abou~ 330 ~g/ml~. The ratio of the ~o DNhs to one ano~her which is op~imum for a par~icular ligase experimen~ was calculated by the method of Du-gaiczyk e~ al~, JMB 96 ~1~75) 1 11 . The incubation was S carried ou~ a. 14C for at least 16 hours.
10. Con~ju~tlon Recipient and donor were cultured overni~h~ to give an OD6~D of 1nO ~ 1r~ (recip;ent) and 1.4 o 1~6 f tdonor)~ and equal volumes of recipient and donor were 10 mixed so ~ha~ the mixture had a large available surface area~ The mix~ure was incuba~ed, w;~hou~ shakin~, a~
37C for ~ hours, and then 20C )~l of it was ~meared c~n- ~
to ayar pla~es, the compos;~ion of wh;ch permitted se~ P
lection a~ainst the donor and For ~he receptor, ~he lat~er bein~J provided with a new prop~r~y ~!hich ~!3S
supplied by the plasrn;d to be transferred. For the 15 conju~a~;on between E. coli HB 1D1 and M. clara DSM
2~7, these were methanol-minimum m~dium plates wi~h the add;t;on of arl antibio~;c tdetermined by the type of the plasnl;d to be transferred)~
In o~her experiments, recipient and donor uere ;ncubated as described above and 2Qû ~ l of this mixture was smeared on~o me~hanol~minimum medium pLates wi~hout anti~io~ic. These plates were incubated at 37C over- i .5 night, and the lawn of bac~eria produced was rinsed off ~ith 2 ml Gf methanol~minimum medium~ 200 ~l of this suspension ~ias then again smeared on me~hanvl~minimum 1-medium plates wi~h the appropria~e an~ibio~ic~ Clones emerged after incubation of the plates at ~7C for r I

, .

- ~.2 - - za 48~72 hours.
11. Expressicn o~ o~i~ DNA in M. clara a) The pLasnlid pBE 3 ~Jas cloned or recloned by the processes described in derivatives of pBR 3~2 which con~ained~ as ~he c~DNA sequence o~ a eukciryo~ic ~ene~ that of monkey insulin.
The monkey insulin c-DNA had been incorpora~ed ;n the Pst I cleavins site of pBR 322 so that this forei0n informa~ion was expressed under ~he con~rol or the ~-lactamase prornotor in E~ coli~ 1h;s produced 2 fusion protein which could be detec~ed with anti insulin an~ibodies ~European Paten~ Applica~ion 0~032J67~.
Monkey insulin c~DNA contains two in~ernal Pst I
cleaving sitesu Thus the coding infornation cannot be removed in an intact forln from ~he plasmid by Ps~ I di gestion. Thus in order ~o make possible replication of th;s plasmid ;n M. clara, the procedure was such tha~
DNA sequences from the M~ clara plasmid pBE 3 ~lere in-corporated in this pBR :~22 derivative expressing insu-~0 l;n information~ The experimen~al procedure was as des-cribed above. By this means, a number o~ hybrid plas-mids were ob~ained which differ ;n their struc~ure frorn the hybrid vec~ors described above onLy by the Inon~
key insulin c~DNA incorpora~ed in ~he pBR 32~ par~
The arrangement of thc insulin gene in ~he pBR 322 part of ~hese vec~ors s~ill remains in phase, so ~ha~ ~hese clones also express insu!in~a~1ti~3enic deter~linan~s in coliO One of ~hese clones, wl-;ch contairls ~he n~ire pBE 3 sequel1ce~ ~as given ~he name pInMc 6~.

21 ~
b~ The rmobilizability defect foul)~ in pB~ 372 can be complemen~ed by plasmids such as CnL K and Col (Youn~ and Poulis~ Gene 4 (1978) 175~17~). Thus, in addi~ion, the plasmi~ Col K was in~roduced by trans~
forma~ion into those donor strains ~i~t) the conju~
ga~ive plasm-id RP ~ and ~he hybrid vec~ors o~ the p~M
series which had been rnade competent by trea~ment with calcium chlor1de and are described above. The colicin~ensi~ive strain AB 1157 was used as the in~
1D dicator strain to detect ~he plasmid Col l~ (~arren e.
al., MGG 170 (1~79) 1D3-107)~ In experin1ei1ts 0l1 cor~Ju gation be~ween the En coli donor s~rains HB 1~1 (RP 4, Col K~ pRM 5~) or HB 101 (RP 4, C~l K, pRM 21) and ~he recipien~ M. clara ATCC 31226~ in order to select ~Of`
clones which contained ~he hybrid ~ector, selection was carriecl ou~t in the presence of high doses of tetracycline ~50 ~g/nll) on me~hanol~mil1irnum n~ed;uln (M 36).
Analys;s of a larye num~er of Mu clara clones showe~ ~hat, in about 10% of ~he case~ clones had be.en obtained which only containod the hybrid vec~or~ :[n- an experirilert on conju0atiorl be~f~eerl the E. coli donor s~rain l-IB 101 (RP 4, Col Kf~ pInMc ~8) and the M. clara recipien~ ATCC 31226, in the salne manner~ M. clara clones were ob~a1ned whicl1 only con~ained the plas m;d pInMc 68.
c~ rhe M. clara clotles wi~h ~he plasmid ~InMc 6S obtain~d ~lere ~hen cultured an~ checked for their in~
sulin conten~ by a radioimmunoassay or a fat-c~ll assay~

Consis~en~ wi~h ~he observa~ior!s made on ~. coli in ~he - 22 ~
~t,'~ European Pa~en~ Application 0,032~675, insulin values be~ween 1 and S IU per liter were also measured in the case o~ M. clara ATCC 312260 Thus the insulin informa-tion contained in ~he pBR 322 par~ of ,he hybrid vector S pInMc 68 is also correc~Lly and ef~ic;ently expressed in M. clarar 12. The s~rain ~,ethylomonas clara ATC~ 31226 is cul-tured in the min-,mum medium M 36 ~o an OD~oo of 0.9 ~o 1.4.
20 ml o~ ~hese cul~ures are introduced in~o 100 ml of M 36 r~edium whicll additionally contains ~% glycine and 10% sucroseO The bacteria are shaken at 100 ~o 180 rpm in this Medium at 37C for 4 hours. No oblong mobile bacteria are ~hen any longer detectable under ~he phase contrast microscope.
1~ Thc spheroplasts produced are cen~ri~-uged down a~ Z~00 x 9 and 4C for 10 minutes and resuspend~d in 1 ml of M 36 medium which additionally contains 10% suc-rose.
The media mentioned can also conta;n, in pla~e of 10~ sucrose, 1 mole per liter of sorbitol or 15%
polyethylene glycol of mean molecular weight 6000.
The spheroplasts can also be separated out by cen~rifu~a~ion at 3~00 x 9 and 4C for 10 m;nu~es.
13a 0~1 ml 0~ a mixture comprising equ~l par~s of resuspension medium and TE buffer and con~aining the plas~id pRM ~1 is added to 0O5 ml of the suspension ob~
tained as in E~ampla 12. 1.5 ml of 4% streng~h poly-~llylene glycol 600D solu~ion 1s added to this mix~ure ~nd i~ ;s carefully and thoYoughly mixed~ rhis mix~lJre z~~ --i5 allo~ed to s~and a~ room tempera~ure for ~ m;nutes~
5 ml of resuspensior1 medium are added and it is centrl--fuged al 3~00 x 9~
The precipitat~ is taken up in 1 ml of resuspen-S sion rnedium and s~reaked on agar pla.es, the compositiol~o~ which csrresponds to the resuspension medium an~,which a~di-tionally con~ain 105% Bacto agar and S0 ~ g/ml of ampi-c;llinD The pla~es are incubated at 37C for 3 days~
and the colonies are cul~ured in M 36 rnediurn which con tains S0~9/ml o~ ampicillin. It ~as possible ~o isolate ~he plasmid p~M 21 employed for transforma~ion from ~the bacteria ~hus obtainedn It is also possible to employ in this process tetracycline at a concentra~ion of 10 ~g/ml in place o~f 1 5 aMpi ci llin.
The same result ;s also ob~a;ned ~hen, in place of the abovelllent;on~!d plasm;d, the plasmid pRM 54 is employed~
14. If the proce~ure is carried ou~ as in Example 13~ but ~he plasmid pInMc 68 is employed and selec~ion is hy means of 50 ~g/ml of ~e~racycline, then Methylo~o-nas clara clones l1aving this plasmid and producing insu~
lin are obtainedn

Claims (57)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A hybrid plasmid with a replicon inherent to an obligate methylotrophic bacterium.

2. A plasmid as claimed in claim 1, which is replicated in a bacterium of the genus Methylomonas.

3. A plasmid as claimed in claim 1, which is replicated in Methylomonas clara.

4. A plasmid as claimed in claim 1, which is replicated in Methylomonas clara ATTC 31226.

5. A plasmid as claimed in claim 1, having integrated eukaryotic DNA.

6. A plasmid as claimed in claim 1, having integrated eukaryo-tic DNA for the expression of insulin.

7. A plasmid as claimed in claim 2, having integrated eukaryo-tic DNA.

8. A plasmid as claimed in claim 2, having integrated eukaryo-tic DNA for the expression of insulin.

9. A plasmid as claimed in claim 1 or 2 having integrated prokaryotic DNA.

10. A process for the preparation of obligate methylotrophic bacteria, which express contained foreign DNA, which comprises a) isolating a plasmid which originates from an obligate methylotropic bacterium and which contains a replicon inherent to the obligate methylotrophic bacterium;

b) preparing from this and from a plasmid with selection markers a hybrid plasmid with a replicon inherent to the obligate methylotrophic bacterium;

c) introducing this hybrid plasmid by transformation into a host organism and amplifying it there;

d) after selection, treating the clones with a suitable conjugative plasmid and abolishing the mobilizability defect;

e) conjugating the clones thus obtained with obligate methylotrophic bacteria as the recipient; and f) selecting the desired clones.

11. The process as claimed in claim 10, wherein the obligate methylotrophic bacteria are of the genus Methylomonas.

12. The process as claimed in claim 10, wherein the obligate methylotrophic bacteria are Methylomonas clara.

13. The process as claimed in claim 10, wherein the recipient is plasmid-free.

14. The process as claimed in claim 13, wherein the recipient is Methylomonas clara ATCC 31 226.

15. The process as claimed in claim 12, wherein the bacterium from which the plasmid is isolated is of the strain DSM 2397.

16; The process as claimed in claim 10, 11 or 12 wherein the plasmid having selection markers may be one or both of pBR 322 and a plasmid containing the genetic informa-tion for the expression of insulin.

17. The process as claimed in claim 13, 14 or 15 wherein the plasmid having selection markers may be one or both of pBR 322 and a plasmid containing the genetic informa-tion for the expression of insulin.

18. The process as claimed in claim 10, 11 or 12 wherein the host organism is Escherichia coli, the conjugative plasmid is RP 4 and the mobilizability defect is abolished by introduction of the plasmid Col K or Col V.

19. The process as claimed in claim 13, 14 or 15, wherein the host organism is Escherichia coli, the conjugative plasmid is RP4 and the mobilizability defect is abolished by introduction of the plasmid Col K or Col V.

20. The process as claimed in claim 10, wherein, in place of the process steps d) and e), the obligate methylotrophic bacterium serving as the recipient is converted into spheroplasts and the hybrid plasmid is inserted by transformation.

21. The process as claimed in claim 11, wherein, in place of the process steps d) and e), the obligate methylotrophic bacterium serving as the recipient is converted into spheroplasts and the hydrid plasmid is inserted by transformation.

22. The process as claimed in claim 12, wherein, in place of the process steps d) and e), the obligate methylotrophic bacterium serving as the recipient is converted into spheroplasts and the hydrid plasmid is inserted by transformation.

23. The process as claimed in claim 13, 14 or 15, wherein in place of the process steps d) and e), the obligate methyl-otrophic bacterium serving as the recipient is converted in-to spheroplasts and the hybrid plasmid is inserted by transfor-mation.

24. The process for the preparation of the spheroplasts of methylotrophic bacteria a mentioned in claim 20, wherein the bacteria are cultured in a glycine-rich medium containing an osmotic stabilizer.

25. The process for the preparation of the spheroplasts of methylotrophic bacteria mentioned in claim 21, wherein the bacteria are cultured in a glycine-rich medium containing an osmotic stabilizer.

26. The process for the preparation of the spheroplasts of methylotrophic bacteria mentioned in claim 22, wherein the bacteria are cultured in a glycine-rich medium containing an osmotic stabilizer.

27. The process as claimed in claim 24, 25, or 26, wherein the glycine-rich medium containing an osmotic stabilizer is slightly hypotonic and containing 2% to 4% by weight of glycine.

28. A host organism containing a plasmid as claimed in claim 1, 2 or 3.

29. A host organism containing a plasmid as claimed in claim 4, 5 or 6.

30. A host organism containing a plasmid as claimed in claim 7 or 8.

31. A host organism containing a plasmid as claimed in claim 1, 2 or 3 and a conjugative plasmid.

32. A host organism containing a plasmid as claimed in claim 4, 5 or 6 and a conjugative plasmid.

33. A host organism containing a plasmid as claimed in claim 7 or 8 and a conjugative plasmid.

34. A host organism containing a plasmid as claimed in claim 1, 2 or 3, a conjugative plasmid and a plasmid abolishing the mobilizability defect.

35. A host organism containing a plasmid as claimed in claim 4, 5 or 6, a conjugative plasmid and a plasmid abolishing the mobilizability defect.

36. A host organism containing a plasmid as claimed in claim 7 or 8, a conjugative plasmid and a plasmid abolishing the mobilizability defect.

37. A host organism containing a plasmid as claimed in claim 1, 2 or 3 and, which contains RP 4 as the conjugative plasmid.

38. A host organism containing a plasmid as claimed in claim 4, 5 or 6 and, which contains RP 4 as the conjugative plasmid.

39. A host organism containing a plasmid as claimed in claim 7 or 8 and, which contains RP 4 as the conjugative plasmid.

40. A host organism containing a plasmid as claimed in claim 1, 2 or 3, RP 4 as the conjugative plasmid and the plasmid Col K or Col V.

41. A host organism containing a plasmid as claimed in claim 4, 5 or 6, RP 4 as the conjugative plasmid and the plasmid Col K or Col V.

42. A host organism containing a plasmid as claimed in claim 7 or 8, RP 4 as the conjugative plasmid and the plasmid Col K or Col V.

43. A spheroplast of a methylotrophic bacterium containing a plasmid as claimed in claim 1, 2 or 3.

44. A spheroplast of a methylotrophic bacterium containing a plasmid as claimed in claim 4, 5 or 6.

45, A spheroplast of a methylotrophic bacterium containing a plasmid as claimed in claim 7 or 8.

46. A spheroplast of an obligate methlotrophic bacterium containing a plasmid as claimed in claim 1, 2 or 3.

47. A spheroplast of an obligate methylotrophic bacterium containing a plasmid as claimed in claim 4, 5 or 6.

48. A spheroplast of an obligate methylotrophic bacterium containing a plasmid as claimed in claim 7 or 8.

49. A spheroplast of the genus methylomonas containing a plasmid as claimed in claim 1, 2 or 3.

50. A spheroplast of the genus methylomonas containing a plasmid as claimed in claim 4, 5 or 6.

51. A spheroplast of the genus methylomonas containing a plasmid as claimed in claim 7 or 8.

52. A spheroplast of Methylomonas clara containing a plasmid as claimed in claim 1, 2 or 3.

53. A spheroplast of Methylomonas clara containing a plasmid as claimed in claim 4, 5 or 6.

54. A spheroplast of Methylomonas clara containing a plasmid as claimed in claim 7 or 8.

55. A spheroplast of Methylomonas clara ATCC 31226 containing a plasmid as claimed in claim 1, 2 or 3.

56. A spheroplast of Methylomonas clara ATCC 31226 containing a plasmid as claimed in claim 4, 5 or 6.

57. A spheroplast of Methylomonas clara ATCC 31226 containing a plasmid as claimed in claim 7 or 8.