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IlllllllllllllIlllllllIllllllllllllllIlllllllllllllllllllllllllllllllllllll US00516976OA United States Patent [19] [11] Patent Number: Wilcox [45] [54] METHOD, VECTORS, AND HOST CELLS FOR THE CONTROL OF EXPRESSION OF HETEROLOGOUS GENES FROM LAC OPERATED PROMOTERS [75] Inventor: Edward R. Wilcox, Escondido, Calif. [73] Assignee: Mycogen Corporation, San Diego, [21] Appl. No.: 386,821 [51] [52] Dec. 8, 1992 Chief, American Society for Microbiology, Washing ton, D.C. Freifelder, D., (1987), “Molecular Biology”, Jones and Bartlett Publishers, Inc., Portola Valley, Calif. Jobe, A., Bourgeois, S., (1972), “The Natural Inducer of the lac Operon”, J. Mol. Biol., 69:397-408. Muller-Hill, H. V. Rickenberger and K. Wallenfels, (1964), “Speci?ecity of the Induction of the Enzymes of the lad Operon in Escherichia coll", J. Mol. Biol., 10:303-318. Calif. [22] Filed: Date of Patent: 5,169,760 Jul. 27, 1989 Primary Examiner-David L. Lacey Int. Cl.5 ..................... .. C12N 15/11; C12N 15/67 US. Cl. .............................. .. 435/691; 435/252.3; 435/320.1; 536/27 [58] Field of Search ................... .. 435/691, 172.3, 39, [56] 435/2523, 320.1; 536/27 References Cited PUBLICATIONS Gene, 51:255-267, 1987, Stark, Multicopy Expression Vectors Carrying the lac Repressor Gene for Regulated High-Level Expression of Genes in Escherichia coli. Beckwith, J ., (1987), “The Lactose Operon", in Escher ichia coli and Salmonella typhimurium Cellular and Mo lecular Biology, vol. 2, Neidhardt, F. C., Editor in Assistant Examiner—John D. Ulm Attorney, Agent, or Firm—-S_aliwanchik 8t Saliwanchik [57] ABSTRACT The invention is a process for controlling the expression of heterologous genes from lac-operated promoters by removing the CAP binding site and lac promoter from the lac operon. Illustrated is the fusion of the lacZ, Y, and A genes of the lac operon to the 3' end of the lacl structural gene. This elimination of the natural regula tory elements of the lac operon results, advantageously, in the production of lac operon gene products in a con stitutive mode from the lac] promoter. 16 Claims, 9 Drawing Sheets U.S. Patent H2ENQ Dec. 8, 1992 2m52E Sheet 1 of 9 2:$3 2;S22‘ . 2; Y gzm ND>IQ J 2m882 T5% 52H32NH2im £2 13HmmomHms>m SWQ2S:5.WH QMX 82 83S2 .@ m mm m2$om3muw222Ez QmEBJI .UE_. US. Patent Dec. 8, 1992 Sheet 2 of 9 5,169,760 H3:moum H22wm 30m oHwmcmw Hwm?mw 2: wHmwm 23N 88ES3: $533$8 88H 2S 28m; , ES8%. ES22 o ow 6E.3 US. Patent HQ3Q8 mUoQHw: Dec. 8, 1992 . :s>m0;. mSN25 Sheet 3 of 9 5,169,760 T32;$5 5%?25ZnwH123 w m o HmZwDHm4:wHu 3%,SSSmPo5m HN:omacx?Em N mm @ . 2;Em6Enus; @2mf?% 8282 US. Patent Dec. 8, 1992 Sheet 4 of 9 o ow 5,169,760 o om mH3w2 o2: 0$ v::2,» Hm H Z E Q azo m :mm: EH23 ON: 2:Guam ‘cw? .558m_96..t522.: co n mH?agm 0$ 8%:m o ow co p .GE0N US. Patent Dec. 8, 1992 Sheet 5 of 9 5,169,760 :3H um o ow vHmgw;m E32:; mg: Hzo m 2EE$8H:2; 2:E?82: S UEQ :25.; UN.GE mg: 83 2HmEXH o ow :$2m. 1 5,169,760 2 Stoneham, MA) is to have enough lac repressor present METHOD, VECTORS, AND HOST CELLS FOR THE CONTROL OF EXPRESSION OF ' HETEROLOGOUS GENES FROM LAC OPERATED PROMOTERS BACKGROUND OF THE INVENTION The lactose (lac) operon consists of three protein products under the control of a lac promoter-operator. These gene products are B-galactosidase (Z), permease (Y), and thiogalactoside transacetylase (A). The protein product of the lad transcript (repressor), an indepen dent gene product, interacts with the operator of the lac operon and keeps synthesis off until allolactose (1,6-0-[3 in the cell, so that transcription from the tac promotor is off until IPTG or another proper inducing B-galacto side is added to the cell. Although IPTG is the current inducer of choice, it is expensive and has been labeled a potential carcinogen. Thus, there is a need to replace IPTG in commercial systems where control of the ex pression of heterologous genes from lac operated pro moters is used. BRIEF SUMMARY OF THE INVENTION The subject invention concerns an improved method for controlling the expression of heterologous genes from lac operated promoters. Speci?cally, the subject D-galactopyranosyl-D-glucose), a product of the B invention concerns a method for controlling the expres galatosidase reaction, accumulates in the cell and binds sion of heterologous genes from lac operated promoters to the repressor. The allolactose repressor complex has which comprises removing the CAP binding site and a changed conformation, allowing the repressor to be lac promoter/operator from the lac operon. The subject displaced from the operator. RNA transcription then invention is exempli?ed herein by use of a novel recom begins from the lac promoter (Beckwith, J. [1987] In 20 binant DNA construct comprising the lacZ, Y, and A Escherichia coli and Salmonella typhimurium Cellular genes of the lac operon, to control the expression of and Molecular Biology. Vol. 2, Neidhardt, F. C., Editor heterologous genes from lac operated promoters. The in Chief, American Society for Microbiology, Washing ton, DC). lacZ, Y, and A genes of the lac operon were fused to the 3’ end of the lacI structural gene, thereby eliminating all A few molecules of the lac operon transcript are 25 of the natural regulatory elements of the lac operon. By present in E. coli, even in the absence of lactose. Hence, doing this, lac operon gene products are produced in a permease and B-galactosidase are always present, at constitutive mode from the lacI promoter and are not least at a low level. Allolactose, the natural inducer of responsive to catabolite repression or to allolactose the operon, is made in the cell when lactose (1,4-O-B-D galactopyanosyl-D-glucose) enters the cell by the per 30 induction. When lactose is added, the constitutively synthesized B-galactosidase converts as much as 20% to mease reaction and is converted through transgalactosi allolactose, which in turn derepresses the tac promoter dation by B-galactosidase into allolactose (Freifelder, and any associated heterologous gene. As long as the D., [1987] Molecular Biology. Jones and Bartlett Publish allolactose concentration in the cell is above 104M, ers, Inc, Portola Valley, CA; Beckwith, supra). It was calculated that greater than 20% of the lactose acted 35 the tac promoter provides high expression .of the pro tein of interest. The complete DNA sequence of the upon by B-galactosidase is converted to allolactose transcriptional fusion of the lad gene to the lacZ, Y, (Jobe, A., Bourgeois, S., [1972] J. Mol. Biol. and A genes of the subject invention is as shown in 69:397-408). The majority of the remaining lactose is converted to glucose and galactose. Allolactose is a Table 1. better substrate for B-galactosidase than lactose (Jobe 40 The novel lacIZYA operon (Table 1) can be inserted in any plasmid, or it can be inserted into any microbial and Bourgeois, supra), and is itself rapidly converted to chromosome to improve control of heterologous gene glucose and galactose. expression from a lac operated promoter (as long as a Another control element of the lac operon is catabo promoter is present to drive lacIZYA transcription). It lite repression. In the presence of glucose the cell is able to repress many operons. For example the lac operon is 45 will be apparent to a person skilled in the art that other constructs can be used to fuse the genes. only transcribed at 2% of its maximum level in the The lac operator, which has very high affinity for the lac] repressor (KDNA=2—5>< 1013 M-l), is a sequence 21 hp long as follows; AATTGTGAGC found to act as a very poor effector molecule. The 50 GGATAACAATT (Barkley, M. D. and S. Bourgeois presence of glucose (Beckwith, supra). When several B-galactosides were compared for their ability to induce the lac operon in vivo, lactose was synthetic non-metabolized B-galactoside, isopropyl-B D-thiogalactoside (IPTG), was found to induce the lactose operon 5 times better than lactose (Monod, J., G. Cohen-Bazire and M. Cohn [1951] Biochirn. Bio phys. Acta. 7:585-599). Yet, allolactose itself is as good 55 an inducer of the operon as IPTG (Muller-Hill, B., H. V. Rickenberg and K. Wallenfels [1964] J. Mol. Biol.‘ 10:303-318; Jobe and Bourgeois, supra). Given the efficiency of lactose conversion to allolac [1978] In The Operon Miller, J. H. and W. S. Reznikoff, eds. Cold Spring Harbor). Many mutations within the lac operator exist which have higher or lower affinities to the lad repressor (Barkley and Bourgeois, supra; Sadler, J. R. et al. [1983] Proc. Natl. Acad. Sci. USA 80:6785-6789; Simons, A. et al. [1984] Proc. Natl. Acad. Sci. USA 81:1624-1628). Many mutations exist within the lad repressor that allow for greater affinity to the lac operator with no significant effect on derepression tose, one might expect lactose to work very well as an (Barkley and Bourgeois, supra). Using any of the many inducer of the lac operon. However, prior to the present disclosure, this has never occurred in practice (Monod et al., supra). The current way to control the expression of heterol different combinations of lac operator and lacI repres sor, the control of heterologous gene expression is ap parent to a person skilled in the art. Many different promoters (eukaryotic as well as prokaryotic) have ogous genes from the lac promoter or lac concensus 65 been placed under lac operator regulation (Yansura, D. promoters such as tac (Rezinkoff, W. S. and W. R. G. and D. J. Henner [1984] Proc. Natl. Acad. Sci. USA McClure [1986] Maximizing Gene Expression, W. Rez 81:439-443; Herrin Jr., G. L. and G. N. Bennett [1984] nikoff and L. Gold, eds., Butterworth Publishers, Gene 321349-356; Deuschle, U., et al. [1986] Proc. Natl. 3 5,169,760 4 HindIII fragment (at the 3' end of the coleopteran active sequence). The 5' end of the tac promoter had been previously inserted by blunt-end ligation into the EcoRI site of pBR322. Acad. Sci. USA 83:4134-4137; Hu, M. C-T. and N. Davidson [1988] Gene 62:301-314; Figge, J., et al. [1988] Cell 52:713-722). The subject invention is de signed to improve lactose induction from such lac oper ated promoters. This invention enables cells to make enough lacZ and DETAILED DISCLOSURE OF THE INVENTION Upon removal of the CAP binding site and lac glucose and galactose, as well as to the true lac inducer, promoter/operator from the lac operon, there is real allolactose. In addition, the invention enables the cell to ized an improved control of expression of a heterolo synthesize enough repressor (lacl protein) to bind to the 10 gous gene. Exempli?ed herein is the insertion of a lacl lac operator upstream from a heterologous gene, and ZYA fusion construct into a plasmid containing a tac Y protein to ef?ciently take up lactose and convert it to keep heterologous gene expression off in the absence of promoted gene encoding a lepidopteran insect toxin. inducer. With excess lactose present in the medium, the cell can accumulate suf?cient concentrations of allolac tose for ef?cient derepression of the lac operator. The plasmid (pMYC47l) was then used to transform Pseudomonas?uorescens, which was designated Pseudo monas?uorescens MR47l (pMYC471). One element of plasmid pMYC47l provides its host BRIEF DESCRIPTION OF THE DRAWINGS with constitutive levels of lac operon gene products. FIG. l-pMYC 2005, LacI and Z operon fusion For this purpose, the operon could be fused to any of a A synthetic fragment of DNA was cloned into variety of available constitutive promoters. In this in pUCl8 to replace the normal sequence found at Hind 20 stance, it was fused to the lacl promoter. In addition, a 111-403 to HaeII-524. This synthetic sequence removes new construct containing a lacIQ promoter, instead of the lac promoter and operator sequences and replaces the lacl promoter, was made (pMYC‘ASS). This new them with a Shine-Dalgarno site. construct provides higher levels of the repressor as well FIGS. 2a to d-pMYC 2101, construction of the lacI as of lac operon gene products. The plasmid, ZYA operon 25 pMYC485, functions like pMYC471. Three fragments of DNA were ligated and then in As a demonstration that lactose inducibility is not troduced by transformation into MC1061 (Casadaban, dependent on the speci?c gene under tac promoter M. and S. Cohen [1980] J. Mol. Biol. 138:179-207) to control, another gene encoding a coleopteran-active construct the plasmid pMYC2lOl. The ?rst fragment came from pMYCZOOS as a HindIII-403 to Eael-485 30 toxin was cloned into the pMYC47l vector replacing the lepidopteran-active toxin gene, resulting in piece, the second fragment came from pMC9 as an pMYCl 161. Pseudomonas?uorescens strain MBlOl car EcoRI-l to EaeI-ll03 piece and the ?nal fragment ryin g this new plasmid also permits lactose induction of comes from pSKS107 as a 9889 bp HindlIl-3l to toxin expression. This will hold true for any gene under EcoRl-l piece. Once constructed, the EcoRI and SalI ends were converted to BamHI and BglII ends, respec 35 control of a lac operated promoter. Another aspect of this fusion is that the lac operon is tively, using oligonucleotide linkers, creating no longer regulated by the normal control elements pMYCZlOl-B. associated with lactose. This means that synthesis of lac FIG. 3 -pMYC467, a tac promoted toxin-containing operon ZYA gene products in these constructs plasmid The tac promoted toxin gene found in pMYC436 40 (pMYC47l, pMYC485, and pMYC1l6l) is constitutive and occurs independently of the concentrations of lac (NRRL deposit no. B-l8292) was cloned as a 4.5 Kbp tose or glucose present in the cell. For example, in the BamHI to PstI fragment in the vector pTJ S260. Not all presence or absence of IPTG or lactose, a new protein the sequence of pTJS260 or of the 3' ?anking sequences band corresponding to the mass of B-galactosidase is of the toxin genes are available, hence some restriction sites are approximated in FIGS. 3, 4, 5 and 6. 45 apparent on SDS-PAGE and the clones cleave X-gal, forming blue colonies under the same conditions. FIG. 4 -pMYC47l, a lactose inducible plasmid For ease in construction, the lacA gene has been The lacIZYA operon was cloned into the BamHI site included on all the plasmids contining lacIZYA men of pMYC467. The resulting plasmid, pMYC471, was tioned in this disclosure. The lacA gene product has no then induced by transformation into MBlOl, a P. ?uo rescens. 50 FIG. 5-pMYC485, an alternate lactose inducible plas mid A lacIQ ZYA operon was constructed by replacing lacl with lacIQ as a 638 bp BamHI to ApaI fragment. role (Freifelder, supra) in production of allolactose and can easily be removed from the lacIZYA operon. For example, there is a nonunique AflII (CTTAAG) restric tion enzyme site a the stop codon of the lacY gene. By using this AflII restriction site, it is possible to remove The lacIQZYA operon was then cloned into the BamHI 55 the lacA gene. The lacIZY operon has the same lactose induction properties as those found in the lacIZYA operon. was tested in M13101 for lactose inducibility. The lacIQ The data of Table 2 demonstrate toxin production is ZYA operon is shown in Table 1 wherein base 16 is a T instead of a C as shown in the table. regulated by lactose or IPTG. Although the amount of lactose used for induction is 10 to 20 fold higher than FIG. 6-pMYCll6l, a coleopteran toxin-lactose in- V site of pMYC467. The resulting plasmid, pMYC485, ducible plasmid IPTG induction levels, the signi?cantly reduced cost of The plasmid pMYC471 was cut with BamHI (at posi tion 12103) and KpnI (at position 7933) to remove the tac-promoted lepidopteran-active toxin and then both ends were ?lled in with T4 DNA polymerase, resulting 65 lactose ($7.00 per kilo, Sigma Chemical Co.) compared to IPTG ($20,000 per kilo, Sigma Chemical Co.) make the former inducer highly economically advantageous. in blunt termini. The tac promoted toxin gene disclosed in US. Pat. No. 4,771,131, was inserted into the above DNA as a blunt-ended Seal (of pBR322) to ?lled-in strate, lactose, must be replenished during cell growth For maximum production levels the metabolized sub or the culture will decrease the synthesis of the tac-pro moted gene. 5,169,760 5 6 380A oligonucleotide synthesizer. This sequence starts Materials and Methods at BP 1073 of the lacl sequence (Farabaugh, P. J ., supra) Cloning and DNA manipulation techniques are de as seen in Table l. Hae 11 Bee 1 CC CGCGGG AAT TTA ACG TGC CAA GT1‘ ACC TGG GCC CGG TCT AGA CCC GGG CGC GCG GCG CGC T'I'G AAC GCC CGG GAT CTA AGA TCT CTG GAC GAA CTT AGC TCG ..Xh_o1 TCA AGT TTA AAT ATG TAC CAA GTT CT C GAG GCA CGT CGA GCT CAG GTC la_cl CAG GTC TGA ACT TCG AGC lacZ Stop‘ 666 CCC GTC CAG S/D GCGCTAGGAGGTAACTI' CGCGATCCTCCATTGAA scribed in Maniatis, T., E. F. Fritsch and J. Sambrook 20 (1982) Molecular Cloning, a Laboratory Manual. Cold Spring Harbor Laboratory Publishers, Cold Spring Harbor, NY. A plasmid containing the lac operon, pSKSl07, was received from Dr. M. Casadaban at the University of Chicago (Shapira, S. K., J. Chou, P. V. Richaud and M. J. Casadaban [1983] Gene 25:71-82). The sequence of the lac operon is published (Kalnins, A., K. Otto, U. Ruther and B. Muller-Hill [1983] EMBO .1. 2:593-597; Hediger, M. A., D. F. Johnson, D. P. Nierlich and I. Zabin [1985] Pro. Nat. Acad. Sci. 82:6414-6418). The lad gene, on a 1.7 KB EcoRl restriction fragment cloned into pBR322, is available from the ATCC, as v‘pMC9, catalogue no. 37195, 37196 and 37197. The se quence of the lac] gene is also published (Farabaugh, P. J. [1978] Nature 274:765-769). The broad host range vector, pMMB22, was received from Dr. M. Bag dasarian (Bagdasarian, M. M., E. Amann, R. Lurz, B. Ruckert and M. Bagdasarian [1983] Gene 26:273-282). A Hindlll fragment of pMMB22,v containing the lacIQ gene, was relinkered to BamHl. The sequence of the lacIQ gene has also been published (Calos, M. P. [1978] Nature 274:762-765). Analysis here is largely based on the published DNA sequences. Some of the critical portions of the constructs were con?rmed by DNA sequencing, e.g., the lacl and laclQ promoters and the fusion region between the lacl transcriptional unit and the lac operon. The vector, pUCl8, is available from a variety of sources, such as catalogue no. 27-4949-01 of Pharmacia Corporation. The broad host range vector, pTJS260, is available from Dr. D. R. Helinski at the University of California, San Diego, La Jolla CA._92093 (Schmidhauser, T. J. [1986] Ph.D Thesis, UCSD). Following are examples which illustrate procedures, including the best mode, for practicing the invention. These examples should not be construed as limiting. All percentages are by weight and all solvent mixture pro portions are by volume unless otherwise noted. EXAMPLE 1 Fusions of the LacI Gene and Lac Operon. Fusions of the lacI gene and lac operon were ?rst carried out in pUCl8. To do so, pUCl8 was linearized with Hindlll and then partially cut with HaeII. The l;1i_nd III Start ATG TAC GAA C l l ICGA The above synthetic sequence removes the PvuII site normally found at BP 1123 of lacI. A new XhoI site was inserted near the 3' end of the lacl gene (BP 1137). These changes were introduced for ease in identi?ca tion of the new construct. No amino acid changes would occur as a result of the mutations introduced in making the base pair changes for the above two restric tion sites. In this construct, the distance between the stop codon of lacl and the start codon of lacZ is 17 base pairs. This region contains a ribosome binding site (marked as S/D) such that a ribosome translating the lacI transcript will be able to continue synthesis of the lacZ gene product from the same transcript. A plasmid diagram of pMYC2005 is seen in FIG. 1. EXAMPLE 2 Con?rmation of Sequence. The synthetic portion of pMYC2005 was sequenced to validate its structure and was used in a three~piece ligation to tie the full lacl gene of pMC9 to the lac operon found in pSKSl07 (FIGS. 20 to d). After trans formation, the lacI/Z fusion region was sequenced from a blue colony on an LB+X-gal (5-bromo-4-chloro-3 indolyl~B-D-galactosidase, a colorimetric substrate for the presence of B-galactosidase) plate. Those restriction sites that were examined yielded fragments of the pre dicted sized (FIGS. 20 to d). The lacl promoter region was also con?rmed by sequence analysis. EXAMPLE 3 Cloning The EcoRI and Sall ends of the lacIZYA construct (FIGS. 2a to d) were respectively relinkered to yield BamHI and BglII restriction sites. The laclZYA operon was cloned into the unique BamHI site of pMYOi67 (FIG. 3) yielding pMYC471 (FIG. 4). The plasmid, pMYC467, contains the tac promoted lepidopteran toxin gene of pMYC436 (a cryIA(c)-like toxin gene [NRRL deposit no. B48292] Adang, M. J. et. al. [1985] Gene 36:289-300) cloned into the broad host range vector, pTJS260. pMYC471 was introduced by trans formation into P. ?ourescens MBlOl and the resulting clone was designated MR471. MR471 was tested for the key elements of the resident pMYC47l plasmid. A func DNA was resolved by electrophoresis on a 1.4% aga rose gel. The 2402 bp band was eluted and ligated to a tional repressor is synthesized because in the absence of double-stranded synthetic DNA insert produced by B-cyanoester chemistry on an Applied Biosystems duced (Table l). The cells are blue when plated on IPTG or lactose no signi?cant amount of toxin is pro X-gal becausea functional B-galactosidase is present. A 5,169,760 11 12 TABLE l-continued 6801 6851 6901 6951 7001 7051 GGCACCAATC AACCGCCG'IT GTGAATACCA CTCGCGGGCG GCGCCACCAG AGGGCGTTAT TCAATATAGA AATCAGACCG CGCGAACCGG CTITGCCACC GCGAGTGTTT GGCGGCACCA ' 710] CACAGCCCCA TCACCAGTGG CGTGCGCTGC TGAAACCGCC GT'ITAATCAC CCGCCTGAAT AGCAGCGCAC CAGCAACAGA GCCAGCTTCC Sall site in pSKSlO7 GTCGAC GATGCCGATC 'I'TTGCGGGTA CT GATGGCGA GGCCAGCGCC CCCACCCCAC AAGCTCACGC CACTGCGACG AGCCCGCCCA CAAGCAGTGC ATCAATGCAC 'ITCGCTGACA TGGTAACCAC 7151 CGAAGCATCA 7201 GCTGCTAAGC 7251 CGACGGCAAT 7301 TGCTGATGAA 7351 CGGCAGAGCG AAGCGGGTAA AAGTAAACAC AGTGGTTGTT ACCAGGCAAA GATACCAGGT AGCCCACCGC CCAGGCAATC CCAGCGTCCA GTCTTGTGGG GAGCGCAACA 'ITCGCTATGT CGCCCATCAG AGGCTGGCGT CGCGCGGGGA AAGAGGCGAC ACGGCAGGCA TGAACTAACC AGCCGCGGAC (l) sequence in bold. between the two ' marks. is synthetic DNA used to fuse the lac operon to the lac l or lac I9 gene operon. (2) pSKSl07 contains an unsequenced mutation at the EcoRl site normally found in the lac Z gene. TABLE 2 Lactose lnducibility of Various Constructs Hours after“) Lactose Toxinm Induction (mM) Cells/ml (ug/ml) 40 40 no lactose 15 24 15 24 23(3) 390) O 2 mM IPTG 20 20 40 40 8.314) 8.314) 24 24 4O 0 24 24 4o 0 MR471 1.6 X 10‘0 2.3 x l0l0 9.1 X to9 1.2 >< 1010 1.8 X 1010 1.1 X 1010 ZYA or laclQZYA operon wherein the CAP binding site and lac promoter/operator is not present in the lac operon. 20 none detected 1003 1011 737 887 1025 1.9 x 1010 2.0 X 1010 6. A transfer vector comprising a lacIZYA or lacIQ' ZYA operon wherein the CAP binding site and lac promoter/operator is not present in the lac operon. 7. A microbial host transformed by the transfer vec tor of claim 6. 25 938 1555 8. The transfer vector, according to claim 6, being a plasmid. MBlOl containing pMYC485 9. A process for preparing a protein product which comprises culturing a microbe comprising a heterolo — - from a lac promoter, and a lacIZYA or lacIQZYA op gous gene encoding said product which is expressed 775 none detected MBlOl containing pMYCl 161 — eron wherein the CAP binding site and lac promoter /operator is not present in the lac operon, until a suffi 300 none detected cient amount of desired product is produced. 10. A process for controlling the expression of a het erologous gene from a lac operated promoter which comprises culturing a microbe comprising a heterolo gous gene expressed from a lac operated promoter, and (“Cultures were induced upon reaching stattonary phase. (“Toxin concentration was determined by laser densitometr) of Coomassie-stained protein bands after electrophoresis of disrupted cells on SDS-PAGE (LKB lnstruc ttonal Manual 222-010). (“This experiment used MR471 grown in a 10L fennentor. All other experimental data were generated using same medium in 250 ml ba?led shake ?asks. min the ferrnentor. 8.3 mM lactose/hour was fed into the culture. It was found that MR4?! did not metabolize this lactose level in a fermentor. resulting in increased concentrations during the experiment. The experiments done in shake flasks were site and lac promoter/operator is not present in the lac gt\ en a single dose of lactose or lPTG at the indicated times. operon. a lacIZY or lacIQZY operon wherein the CAP binding 11. The process, according to claim 10, wherein said microbe has been transformed with a plasmid compris ing a heterologous gene. 12. The process according to claim 10, wherein said ability to control expression of heterologous genes from 45 microbe comprises a chromosome comprising said lac operated promoters as does the parent operon. lacIZY or lacIQZY operon wherein the CAP binding 2. A novel laclQ ZYA operon having the sequence site and lac promoter/operator is not present in the lac I claim: 1. A novel lacl ZYA operon having the sequence shown in Table l, or mutations thereof which retain the shown in Table 1, wherein base 16 is a T instead of a C as shown in the table, or mutations thereof which retain operon. 13. A transfer vector comprising a lacIZY or la the ability to control expression of heterologous genes cIQZY operon wherein the CAP binding site and lac promoter/operator is not present in the lac operon. 14. A microbial host transformed by the transfer vec from lac operated promoters as does the parentoperon. 3. A process for controlling the expression of a heter ologous gene from a lac operated promoter which com tor of claim 13. prises culturing a microbe comprising a heterologous 15. The transfer vector, according to claim 13, being gene expressed from a lac operated promoter, and a 55 a plasmid. lacIZYA or lacIQZYA operon wherein the CAP bind 16. A process for preparing a protein product which ing site and lac promoter/operator is not present in the comprises culturing a microbe comprising a heterolo lac operon. gous gene encoding said product which is expressed 4. The process, according to claim 3, wherein said from a lac promoter, and a laclZY or lacIQZY operon microbe has been transformed with a plasmid compris 60 wherein the CAP binding site and lac promoter/opera ing the heterologous gene. ‘tor is not present in the lac operon, until a sufficient 5. The process according'to claim 3, wherein said amount of desired product is produced. microbe comprises a chromosome comprising a lacl it 65 t it t it