# Patent

## Title
Continuous microorganism cultivating apparatus

## Abstract

Some of the work disclosed herein was supported by an NIH Support Grant number NIH-5-R01-AI03038.

## Background
Many proteins of industrial and medical interest can effectively be produced by introducing the corresponding genes into microorganisms using genetic engineering techniques. A favorite microorganism for such production is Escherichia coli, whose genetic makeup is probably the best known of all organisms, and whose mutants lend themselves to elaborate manipulations.

Production of a protein in E. coli can yield very large amounts of protein, which must usually be purified to remove other proteins and bacterial components (including bacterial endotoxin).

It is desirable to devise a system such that the cells of E. coli will produce a desired protein and release it into the medium, thereby facilitating purification. With such a system, it may also be possible to collect secreted proteins in large amounts from E. coli cells growing under continuous flow conditions.

The kil gene is identified as an open frame in plasmid pColE1,the source of colicin El. The MIT group (Drs. S.E. Luria and J.L. Suit) have demonstrated the existence and the role of the kil gene: Expression of an intact kil gene located under the ColE1 promoter causes cell death and release of colicin El from the bacteria (Suit, et al. [1983]J. Bacteriol. 161:944-948). Colicin El, like all colicins, is produced in the cytoplasm, and is not located in the periplasm. Several other colicin-producing plasmids have been found to contain genes which function analogously to that of kil in pCo1E1 (Sabik, J.F., Suit, J.L. and Luria, S.E. [1983]J. Bacteriol. 153:1479-1485. The colicin-kil promoters in all these plasmids are inducible by treatment with mitomycin C. The protein product of kil has not yet been isolated; it is believed to be made as a peptide of 45 amino acids, which may be processed in vivo by proteolysis.

When the kil gene is expressed under a strong promoter, kil.sup.s cells of E. coli are rapidly killed. Recently Kobayashi et al. (Kobayashi, T. et al. [1986]J. Bacteriol. 166:728-732) reported that weak expression of kil (under an unknown weak promoter) causes release of two periplasmic proteins (.beta.-lactamase and alkaline phosphatase) with little cell killing.

Recently, researchers at MIT published on the "Expression of the Cloned ColE1 kil Gene in Normal and Kil.sup.r Escherichia coli, " (Altieri, M., Suit. J.L., Fan, M.-L.J. and Luria, S.E. [1986]J. Bacteriol. 168:648-654). This paper makes no mention of the ability of the prepared mutants to release periplasmic proteins into the medium. (Kobayashi et al., supra).

Utilizing a novel selection process, novel and useful mutant microorganisms were made. These novel microorganisms are mutants of E. coli K12 A153 NRRL B-18164). These novel mutants are representative of two distinct classes possessing the common features of being resistant to the lysing action of the kil gene of ColE1 and pColE2,  and, when carrying an expressed Kil gene, of being capable of releasing, advantageously, substantial amounts of any of several periplasmic E. coli or recombinant proteins into the culture medium. The specific mutants exemplified herein are prototypes of two classes of mutants which can be prepared by the novel process dislcosed herein. Mutants designated Class I release periplasmic proteins in amounts of about 20 to about 50% throughout the growth cycle, whereas mutants belonging to Class II release up to about 100% of periplasmic proteins only when they approach full growth in the culturing process.

FIG. 1 Construction of plasmids pBLK1, pUK1L , and pSEL5. Abbreviations and symbols: (.fwdarw.) recognition site for HaeIII restriction endonuclease: p(BamHI).sub.8 and p(EcoRI).sub.8, 8-bp linkers containing the BamHI and EcoRI recognition sequences, respectively; ligase, T4 DNA ligase; BAP, bacterial alkaline phosphatase; kil, an opening reading frame of 138 bp on pCo1E1 containing a unique NruI recognition sequence; Ap.sup.r and Tc.sup.r, genes coding for resistance to ampicillin and tetracycline, respectively; lac, the proximal part of the lactose operon including promoter (p) and operator (o) regions, plus a 30-bp insert containing the indicated restriction endonuclease recognition sequences and the first few base pairs of the gene coding for B-galactosidase(Z).

The following deposits of cultures disclosed in this application have been made in the Agricultural Research Culture Collection (NRRL), Northern Regional Research Center, U.S. Department of Agriculture, 1815 North University Street, Peoria, Ill. 61604, USA Culture         Repository No. Deposit Date ______________________________________ E. coli K12 A153 NRRL B-18164 Jan. 30, 1987 E. coli K12 A153 NRRL B-18165 Jan. 30, 1987 kil.sup.R I-1(pUK1L) E. coli K12 A153 NRRL B-18166 Jan. 30, 1987 kil.sup.R II-1(pUK1L) E. coli K12 A153 NRRL B-18167 Jan. 30, 1987 kil.sup.R I-1(pUK1L) (pAOmpA) ______________________________________

The subject cultures have been deposited under conditions that assure that access to the cultures will be available during the pendency of this patent application to one determined by the Commissioner of Patents and Trademarks to be entitled thereto under.37 CFR 1.14  and 35 USC 122. The deposits are available as required by foreign patent laws in countries wherein counterparts of the subject application, or its progeny, are filed. However, it should be understood that the availability of a deposit does not constitute a license to practice the subject invention in derogation of patent rights granted by governmental action.

Further, the subject culture deposits will be stored and made available to the public in accord with the provisions of the Budapest Treaty for the Deposit of microorganisms, i.e., they will be stored with all the care necessary to keep them viable and uncontaminated for a period of at least five years after the most recent request for the furnishing of a sample of the deposit, and in any case, for a period of at least 30 (thirty) years after the date of deposit or for the enforceable life of any patent which may issue disclosing the cultures. The depositor acknowledges the duty to replace the deposits should the depository be unable to furnish a sample when requested, due to the condition of the deposits. All restrictions on the availability to the public of the subject culture deposits will be irrevocably removed upon the granting of a patent disclosing them.

The expression system described herein using healthy Kil.sup.r mutants and the kil gene to release periplasmic proteins into the culture medium is of great utility in producing proteins of medical and industrial importance. The advantage of this system is that the desired protein should be easier to purify from other bacterial proteins and bacterial endotoxins. In addition, the desired protein can be produced under continuous culture conditions. Proteins of mammalian or plant origin engineered to be periplasmic in bacteria will also be released. Examples of mammalian proteins that have been placed in the bacterial periplasm include human epidermal growth factor (Oka, T. et al. [1985]Proc. Natl. Acad. Sci. 82:7212-7217) and human interferon alpha 2 (Barbero, J.L. et al. [1986]J. of Biotechnology 4: 255-267). Some important plant proteins, including ribulose diphosphate carboxylase and leguminous hemoglolin, have also been cloned into E. coli. The release of these periplasmic proteins into the culture medium will be of great utility. Examples of other proteins that can be produced using the invention system include, but are not limited to, medically important proteins such as IL-1 and HTLV-III proteins, and industrial enzymes such as ligninases and glucanases.

Bacterial hosts other than E. coli can be used in the subject invention by transferring the kil gene, either in pUK1L or pCo1E1 plasmid, to Kil.sup.r mutants such as Salmonella or Klebsiella. Further, the kil gene can be transferred to Rhizobium by mating. The procedures for transferring the kil gene are well known to persons skilled in the art.

The kil gene also can be transferred by making cointegrates of pUK1L with plasmids such as R300B (incompatibility iQ group) that can enter by mating into Pseudomonas and other organisms.(See Barth, Tobin, and Sharpe, Development of broad host-range plasmid vectors, in Molecular Biology, Pathogenicity, and Ecology of Bacterial Plasmids, S.B. Levy, R.C. Clowes, and E.L. Koenig, eds. New York, Plenum, 1981, pp.439-443.)

The construction of plasmids pBK1, pUK1L, and pSEL5 is outlined in FIG. 1. The probable site of the kil gene of pColE1 is downstream from the cea gene, very near the imm gene (Sabik, J.F., J.L. Suit, S.E. Luria [1983] "cea-kil operon of the ColE1 plasmid," J. Bacteriol. 153:1479-1485; Zhang, S., A. Faro, G. Zubay [1985] "Mitomycin-induced lethality in Escherichia coli cells continuing the ColE1 plasmid: involvement of the kil gene," J. Bacteriol. 163:174-179). In this region there is an open reading frame of 138 bp, lying in a 412-bp DNA fragment bracketed by two Haelll sites and containing the only Nrul site on the ColE1 plasmid (Chan, P.T., H. Ohmori, J. Tomazawa, J. Lebowitz [1985] "Nucleotide sequence organization of ColE1 DNA," J. Biol. Chem. 260:8925-8935; Dougan, G., M. Saul, G. Warren, D. Sherrat [1978] "A functional map of plasmid ColE1," Mol. Gen. Genet. 148:325-327; Oka, A., N. Nomura, M. Morita, H. Sugisaki, K. Sugimoto, M. Takanami [1979] "Nucleotide sequence of small ColE1 derivatives: structure of the regions essential for autonomous replication and colicin E1 immunity," Mol. Gen. Genet. 172:151-159). DNA fragments approximately 400 bp long produced by Haelll digestion of pColE1 were cloned into the unique BamHl site of pBR322. The desired product was identified as a plasmid conferring resistance to ampicillin but not tetracycline and containing two Nrul sites and was called pBK1. The inserted fragment presumed to contain the kil gene was isolated from a BamHl digest of pBK1 and cloned into the unique BamHl site of the plasmid pUR222, which contains the lac promoter and the first part of the IacZ gene as well as an Ap.sup.r marker. Recombinant plasmids with DNA inserted into the cloning site gave rise to Ap.sup.r transformants of E. coli RRl.increment.M15 that were white on LB azar plates containing 5-bromo-4-chloro-3-indolyl-.beta. -D -galactopyranoside an indicator (Ruther, U., M. Koenen, K. Otto, B. Muller-Hill [1981] "pUR22 a vector for cloning and rapid chemical sequencing of DNA," Nucleic Acids Res. 9:4087-4098). Plasmids were isolated from such transformed cells, and those that released a 300 bp fragment upon double digestion with Nrul and EcoRl were considered to be pUR222 derivatives in which the kil gene DNA fragment has been inserted in the direction of transcription of the lac promoter. One such plasmid was then called pUK1L.

Plasmid pSEL5 was constructed by inserting a synthetic EcoRl linker into the Nrul site in the kil gene and was identified as an Ap.sup.r plasmid that was nonlethal to strain A153. Restriction enzyme tests showed it to have two EcoRl sites 300 bp apart and no Nrul site.

Following are examples which illustrate procedures for practicing the invention. These examples should not be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.

E. coli K12 A153 cells were subjected to genetic manipulations as follows: Cells of A153 were transformed with plasmid DNA, that of either pDMS630 (available from Dartmouth Medical School) and having the following relevant features: pColE1::Tn3(Ap.sup.r, a that is, a kil gene together with a gene for ampicillin resistance or cea.sup.- deletion derivatives of it, and 22 individual Ap.sup.r transformants were subjected to several successive cycles of treatment with mitomycin C. For each cycle of treatment, an overnight culture was diluted 1/50 and incubated at 37.degree. C with shaking for about 3 h and then with mitomycin C (2 .mu.g/ml) for 2 h, at which time survival was 0.01 to 0.1%. The culture was then diluted 10.sup.-5 and incubated overnight. After 3 to 5 such cycles, cultures were streaked out for isolated colonies on LB-ampicillin plates. (LB medium: 10 g/l bacto-tryptone, 5 g/l bacto-yeast extract, 10 g/l NaCl, pH 7.5). Five colonies were picked from each plate, grown up, treated for 2 h with mitomycin C, and tested by scoring for the time of appearance of visible growth. Cultures that showed greater survival than the parent control were retained and rechecked by measuring culture turbidity at intervals after the addition of mitomycin C. Those that did not show the loss of turbidity typical for mitomycin C-induced cells with kil.sup.+ plasmids were the putative Kil-resistant (Kil.sup.r) mutants. Each putative Kil.sup.r isolate was put through the usual plasmid DNA amplification and extraction procedures. When plasmid DNA was found, it was used to transform A153 cells to Ap.sup.r, and the transformants were checked for normal kil.sup.+ expression upon mitomycin C induction. Of 37 putative resistant mutants tested in this way, 34 were found to contain a kil.sup.+ plasmid.

Other compounds which can be used in the selection medium in place of ampicillin are those for which a resistance gene is found on the plasmid containing the kil gene. For example, the plasmid can contain a tetracycline resistance gene, which would then require the presence of tetracycline in the selection medium.

Twelve mutants were screened for sensitivity or resistance to the following agents thought likely to detect changed membrane properties: methylene blue, acriflavine, EDTA, neomycin, and deoxycholate. The screen used LB plates into which the chemicals had been incorporated at various concentrations, and the plates were scored for growth after incubation at 30.degree. C. The mutants fell into three classes: class I (one mutant), sensitive to EDTA, neomycin, and deoxycholate (no growth at concentrations of 2 mM, 1 .mu.g/ml, and 1%, respectively): class II (eight mutants) resembling the control strain (growth at concentrations of 5 mM, 2 .mu.g/ml, and 3%, respectively): and class III (three mutants) resistant to 6 .mu.g of neomycin per ml. The response to methylene blue or to acriflavine was normal in all isolates.

One representative of each of these classes was cured of its resident plasmid. The cured strains were designated I-1, II-1, and III-1, respectively, and were studied further. Each was found to display the same response to EDTA, neomycin, and deoxycholate as before being cured. The growth of mutant II-1 was found to be slow at temperatures above 35.degree. C, so all incubations were carried out at that or lower temperatures. Mutant III-1 grew somewhat more slowly than the parent and the other mutants at all temperatures.

The above procedure is disclosed in Altieri et al., supra. This publication is incorporated herein by reference thereto.

Mutants Kil.sup.r I-1 and II-1 are prototypes of two classes of mutants isolated in the above way. Many such mutants, Kil.sup.r I and Kil.sup.r II, can be obtained by the above procedure.

The Kil.sup.r I-1 and II-1 strains were cured of the pColE1 plasmid and individual cultures of cured cells were transformed with plasmid pUK1L , constructed as shown in Altieri, suora, in which the kil gene from pColE1 is placed under the promoter-operator of the lac operon. The cells have no lac repressor; hence the kil gene is maximally expressed. Both Kil.sup.r I-1 and Kil.sup.r II-1 cells were fully resistant to kil expression.

The cells of Kil.sup.r I-1 (pUK1L) released 20 to 50% of their periplasmic enzymes B-lactamase and alkaline phosphatase throughout their growth. Cells of Kil.sup.r II-1 (pUK1L) only release periplasmic enzymes (50-100%) in aging cultures.

The role of mutations I and II in release of protein is specific. A third class of mutants, Kil.sup.r III, does not release any significant amounts of periplasmic proteins.

The protein A gene from Staphylococcus aureus was cloned behind the E. coli ompA signal sequence. First, EcoRI linkers were ligated to a 1.1 kb Fnu4HI fragment containing the protein coding sequence of protein A. Second, the fragment with EcoRI ends was ligated into the EcoRI site of the secretion cloning vector pIN-III-ompA (Ghrayeb, J. et al. [1984]EMBO Journal 3:2437-2442). Finally, this plasmid was made chloramphenicol-resistant and ampicillin-sensitive by inserting the CAT (chloramphenicol acyltransferase) gene into the bla (.beta.-lactamase) gene. The resultant plasmid, pAompA, conferred chloramphenicol resistance and directed the production and location of protein A into the periplasmic space. Protein A can be recovered from the periplasmic space by the same well-known procedures by which one can selectively extract .beta.-lactamase and alkaline phosphatase (but none of the cytoplasmic enzymes). In the absence of a Kil gene, cells with plasmid pAOmpA do not release any of the periplasmic proteins into the medium.

Tables 1-4 illustrate typical findings of enzyme secretion of periplasmic enzymes, as well as the failure of secretion when the kil gene is inactive. They also show that cells secreting periplasmic enzymes do not secrete significant amounts of a cytoplasmic enzyme, .beta.-galactosidase, indicating that protein release is not a simple consequence of cell lysis. __________________________________________________________________________ Release of Enzymes from Escherichia coli A153 I-1 Carrying __________________________________________________________________________ pUK1L .beta.-lactamase .beta.-D-galactosidase A. Plasmid Sample Total Free % Free Total Free % Free __________________________________________________________________________ pUK1L 1   --  32 --   1.23 0.007 0.6 2   200 128 .about.50 3.38 0.235 1.1 3   800 512 60   37.6 0.1 0.3 pSEL5 1   100  4  4   1.17 0.008 0.48 2   200 16  8   9.8  0.007 0.08 3   800 16  2   45.0 0.008 0.02 __________________________________________________________________________ alkaline phosphatase B.     Total Free % Free __________________________________________________________________________ pUK1L 0.032 0.007 21.00 PSEL5 0.048 0.001 0.02 __________________________________________________________________________ ______________________________________ Release of .beta.-lactamase from Kil.sup.r Mutants Carrying pUKlL .beta.-Lactamase Mutant    Plasmid  Total      Free % Free ______________________________________ II-l      pUK1L    l28        l28  l00 pSEL5    256         8    3 III-l     pUKlL     64         4    6 pSEL5     64         4    6 ______________________________________

Methods are as given in Table 1. The bacteria were taken from overnight cultures. .beta.-lactamase titers are in empirical units from 1:2 serial dilutions. ______________________________________ Release of Enzymes from Escherichia coli W31101.sup.q (pUK1L) Sam-     .beta.-lactamase .beta.-D-galactosidase Plasmid ple    Total   Free % Free Total Free % Free ______________________________________ pUK1L a       400     200  50     43   2.1  5.0 b      1600    1600 100    217   7.8  3.4 pSEL5 a       400     40   10    113   2.6  2.3 b      1600     80   5     240   2.4  1.0 ______________________________________

Cultures of W31101.sup.q carrying either pUK1L or pSEL5 were grown first with isopropyl-.beta.-D-thiogalactopyranoside (IPTG, Sigma Chemical, St. Louis, MO) (0.02 mM) to induce expression of the kil gene and of .beta.-D-galactosidase (samples a), then for 2 h without IPTG (samples b). Assay was done as in Table 1. ______________________________________ Qualitative Estimate of Hybrid Protein A Released A153 I-1 Al53 I-1 (pAOmpA) (pAOmpA, pUK1L) Strain   Cells  Medium   Wash  Cells Medium Wash ______________________________________ Relative 99%    1%       --    80%  7%     13% Amount Hybrid Protein A ______________________________________

Cells of Escherichia coli A153 I-1 (pAOmpA) with or without pUK1L were grown at 30.degree. C in LB broth overnight. Maintenance of both plasmids required a double drug selection. A153 I-1 (pAOmpA) was grown in LB broth with 10 .mu.g/ml chloramphenicol; A153 I-1 (pAOmpA: pUK1L) with 10 .mu.g/ml chloramphenicol and 50 .mu.g/ml ampicillin. Culture samples were centrifuged to remove medium from cells and the cells were washed once with 10 mM Tris pH 8.0, 1 mM EDTA for 10 min and recentrifuged to separate cells from wash. Fractionated culture samples were assayed for relative amounts of hybrid protein A by Western analysis (Maniatis, T., Fritsch, E.F. and Sambrook, J., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY [1982]) Protein A was visualized by reaction with rabbit IgG conjugated to horseradish peroxidase (Sigma). 4-chloro-1-naphthol was used as the substrate. Serial dilutions of fractionated culture samples allowed a relative estimation of the amount of hybrid protein A in each fraction.

## Claims

1. E. coli mutant microbes, designated Class I, characterized as follows: such gene with kil-type activity; E. coli or recombinant proteins into the culture medium when carrying an expressed kil gene or other gene with kil-type activity; and being further characterized as about 50% throughout the growth cycle.

2. E. coli mutant microbes, designated Class II, characterized as follows: such gene with kil-type activity; E. coli or recombinant proteins into the culture medium when carrying an expressed kil gene or other gene with kil-type activity; and being further characterized as about 100% only when they approach full growth in the culturing process.

3. E. coli K12 A153 kil.sup..RTM. I-1(pUKIL), a microbe according to claim 1.

4. E. coli K12 A153 kil.sup.2.RTM. II-1(pUKIL), a microbe according to claim 2.

5. E. coli K12 A153 kil.sup..RTM. I-1(pUKIL)(pAOmpA), a microbe according to claim 1.

6. The E. coli, according to claim 1, wherein said other gene with kil-type activity is the kil-type gene of pCo1E2.

7. The E. coli, according to claim 2, wherein said other gene with kil-type activity is the kil-type gene of pCo1E2.