Method, vectors, and host cells for the control of expression of heterologous genes from lac operated promoters

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 lacI structural gene. This elimination of the natural regulatory elements of the lac operon results, advantageously, in the production of lac operon gene products in a constitutive mode from the lacI promoter.

Description

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 .beta.-galactosidase (Z), permease (Y), and thiogalactoside transacetylase (A). The protein product of the lacI transcript (repressor), an independent gene product, interacts with the operator of the lac operon and keeps synthesis off until allolactose (1,6-0-.beta.-D-galactopyranosyl-D-glucose), a product of the .beta.-galatosidase reaction, accumulates in the cell and binds to the repressor. The allolactose repressor complex has a changed conformation, allowing the repressor to be displaced from the operator. RNA transcription then begins from the lac promoter (Beckwith, J. [1987] In Escherichia coli and Salmonella typhimurium Cellular and Molecular Biology. Vol. 2, Neidhardt, F. C., Editor in Chief, American Society for Microbiology, Washington, D.C.).

A few molecules of the lac operon transcript are present in E. coli, even in the absence of lactose. Hence, permease and .beta.-galactosidase are always present, at least at a low level. Allolactose, the natural inducer of the operon, is made in the cell when lactose (1,4-0-.beta.-D-galactopyanosyl-D-glucose) enters the cell by the permease reaction and is converted through transgalactosidation by .beta.-galactosidase into allolactose (Freifelder, D., [1987] Molecular Biology. Jones and Bartlett Publishers, Inc., Portola Valley, CA; Beckwith, supra). It was calculated that greater than 20% of the lactose acted upon by .beta.-galactosidase is converted to allolactose (Jobe, A., Bourgeois, S., [1972] J. Mol. Biol. 69:397-408). The majority of the remaining lactose is converted to glucose and galactose. Allolactose is a better substrate for .beta.-galactosidase than lactose (Jobe and Bourgeois, supra), and is itself rapidly converted to glucose and galactose.

Another control element of the lac operon is catabolite repression. In the presence of glucose the cell is able to repress many operons. For example the lac operon is only transcribed at 2% of its maximum level in the presence of glucose (Beckwith, supra).

When several .beta.-galactosides were compared for their ability to induce the lac operon in vivo, lactose was found to act as a very poor effector molecule. The synthetic non-metabolized .beta.-galactoside, isopropyl-.beta.-D-thiogalactoside (IPTG), was found to induce the lactose operon 5 times better than lactose (Monod, J., G. Cohen-Bazire and M. Cohn [1951] Biochim. Biophys. Acta. 7:585-599). Yet, allolactose itself is as good 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 allolactose, one might expect lactose to work very well as an 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 heterologous genes from the lac promoter or lac concensus promoters such as tac (Rezinkoff, W. S. and W. R. McClure [1986] Maximizing Gene Expression, W. Reznikoff and L. Gold, eds., Butterworth Publishers, Stoneham, MA) is to have enough lac repressor present in the cell, so that transcription from the tac promotor is off until IPTG or another proper inducing .beta.-galactoside 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 expression of heterologous genes from lac operated promoters 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. Specifically, the subject invention concerns a method for controlling the expression of heterologous genes from lac operated promoters which comprises removing the CAP binding site and lac promoter/operator from the lac operon. The subject invention is exemplified herein by use of a novel recombinant DNA construct comprising the lacZ, Y, and A genes of the lac operon, to control the expression of heterologous genes from lac operated promoters. The lacZ, Y, and A genes of the lac operon were fused to the 3' end of the lacI structural gene, thereby eliminating all of the natural regulatory elements of the lac operon. By doing this, lac operon gene products are produced in a constitutive mode from the lacI promoter and are not responsive to catabolite repression or to allolactose induction. When lactose is added, the constitutively synthesized .beta.-galactosidase converts as much as 20% to allolactose, which in turn derepresses the tac promoter and any associated heterologous gene. As long as the allolactose concentration in the cell is above 10.sup.-5 M, the tac promoter provides high expression of the protein of interest. The complete DNA sequence of the transcriptional fusion of the lacI gene to the lacZ, Y, and A genes of the subject invention is as shown in Table 1.

The novel lacIZYA operon (Table 1) can be inserted in any plasmid, or it can be inserted into any microbial chromosome to improve control of heterologous gene expression from a lac operated promoter (as long as a promoter is present to drive lacIZYA transcription). It will be apparent to a person skilled in the art that other constructs can be used to fuse the genes.

The lac operator, which has very high affinity for the lacI repressor (K.sub.DNA =2-5.times.10.sup.13 M.sup.-1), is a sequence 21 b.p. long as follows; AATTGTGAGCGGATAACAATT (Barkley, M. D. and S. Bourgeois [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 lacI 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 lacI repressor that allow for greater affinity to the lac operator with no significant effect on derepression (Barkley and Bourgeois, supra). Using any of the many different combinations of lac operator and lacI repressor, the control of heterologous gene expression is apparent to a person skilled in the art. Many different promoters (eukaryotic as well as prokaryotic) have been placed under lac operator regulation (Yansura, D. G. and D. J. Henner [1984] Proc. Natl. Acad. Sci. USA 81:439-443; Herrin Jr., G. L. and G. N. Bennett [1984] Gene 32:349-356; Deuschle, U., et al. [1986] Proc. Natl. 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 designed to improve lactose induction from such lac operated promoters.

This invention enables cells to make enough lacZ and Y protein to efficiently take up lactose and convert it to glucose and galactose, as well as to the true lac inducer, allolactose. In addition, the invention enables the cell to synthesize enough repressor (lacI protein) to bind to the lac operator upstream from a heterologous gene, and keep heterologous gene expression off in the absence of inducer. With excess lactose present in the medium, the cell can accumulate sufficient concentrations of allolactose for efficient derepression of the lac operator.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1-pMYC 2005, LacI and Z operon fusion

A synthetic fragment of DNA was cloned into pUC18 to replace the normal sequence found at Hind III-403 to HaeII-524. This synthetic sequence removes the lac promoter and operator sequences and replaces them with a Shine-Dalgarno site.

FIGS. 2a to d-pMYC 2101, construction of the lacIZYA operon

Three fragments of DNA were ligated and then introduced by transformation into MC1061 (Casadaban, M. and S. Cohen [1980] J. Mol. Biol. 138:179-207) to construct the plasmid pMYC2101. The first fragment came from pMYC2005 as a HindIII-403 to EaeI-485 piece, the second fragment came from pMC9 as an EcoRI-1 to EaeI-1103 piece and the final fragment comes from pSKS107 as a 9889 bp HindIII-31 to EcoRI-1 piece. Once constructed, the EcoRI and SalI ends were converted to BamHI and BglII ends, respectively, using oligonucleotide linkers, creating pMYC2101-B.

FIG. 3 -pMYC467, a tac promoted toxin-containing plasmid

The tac promoted toxin gene found in pMYC436 (NRRL deposit no. B-18292) was cloned as a 4.5 Kbp BamHI to PstI fragment in the vector pTJS260. Not all the sequence of pTJS260 or of the 3' flanking sequences of the toxin genes are available, hence some restriction sites are approximated in FIGS. 3, 4, 5 and 6.

FIG. 4 -pMYC471, a lactose inducible plasmid

The lacIZYA operon was cloned into the BamHI site of pMYC467. The resulting plasmid, pMYC471, was then induced by transformation into MB101, a P. fluorescens.

FIG. 5-pMYC485, an alternate lactose inducible plasmid

A lacI.sup.Q ZYA operon was constructed by replacing lacI with lacI.sup.Q as a 638 bp BamHI to ApaI fragment. The lacI.sup.Q ZYA operon was then cloned into the BamHI site of pMYC467. The resulting plasmid, pMYC485, was tested in MB101 for lactose inducibility. The lacI.sup.Q ZYA operon is shown in Table 1 wherein base 16 is a T instead of a C as shown in the table.

FIG. 6-pMYC1161, a coleopteran toxin-lactose inducible plasmid

The plasmid pMYC471 was cut with BamHI (at position 12103) and KpnI (at position 7933) to remove the tac-promoted lepidopteran-active toxin and then both ends were filled in with T4 DNA polymerase, resulting in blunt termini. The tac promoted toxin gene disclosed in U.S. Pat. No. 4,771,131, was inserted into the above DNA as a blunt-ended ScaI (of pBR322) to filled-in 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.

DETAILED DISCLOSURE OF THE INVENTION

Upon removal of the CAP binding site and lac promoter/operator from the lacoperon, there is realized an improved control of expression of a heterologous gene. Exemplified herein is the insertion of a lacIZYA fusionconstruct into a plasmid containing a tac-promoted gene encoding a lepidopteran insect toxin. The plasmid (pMYC471) was then used to transform Pseudomonas fluorescens, which was designated Pseudomonas fluorescens MR471 (pMYC471).

One element of plasmid pMYC471 provides its host with constitutive levels of lac operon gene products. For this purpose, the operon could be fused to any of a variety of available constitutive promoters. In this instance,it was fused to the lacI promoter. In addition, a new construct containing a lacI.sup.Q promoter, instead of the lacI promoter, was made (pMYC485). This new construct provides higher levels of the repressor as well as of lac operon gene products. The plasmid, pMYC485, functions like pMYC471.

As a demonstration that lactose inducibility is not dependent on the specific gene under tac promoter control, another gene encoding a coleopteran-active toxin was cloned into the pMYC471 vector replacing the lepidopteran-active toxin gene, resulting in pMYC1161. Pseudomonas fluorescens strain MB101 carrying this new plasmid also permits lactose induction of toxin expression. This will hold true for any gene under control of a lac operated promoter.

Another aspect of this fusion is that the lac operon is no longer regulatedby the normal control elements associated with lactose. This means that synthesis of lac operon ZYA gene products in these constructs (pMYC471, pMYC485, and pMYC1161) is constitutive and occurs independently of the concentrations of lactose or glucose present in the cell. For example, in the presence or absence of IPTG or lactose, a new protein band corresponding to the mass of .beta.-galactosidase is apparent on SDS-PAGE and the clones cleave X-gal, forming blue colonies under the same conditions.

For ease in construction, the lacA gene has been included on all the plasmids contining lacIZYA mentioned in this disclosure. The lacA gene product has no role (Freifelder, supra) in production of allolactose and can easily be removed from the lacIZYA operon. For example, there is a nonunique AflII (CTTAAG) restriction enzyme site a the stop codon of the lacY gene. By using this AflII restriction site, it is possible to remove the lacA gene. The lacIZY operon has the same lactose induction propertiesas those found in the lacIZYA operon.

The data of Table 2 demonstrate toxin production is regulated by lactose orIPTG. Although the amount of lactose used for induction is 10 to 20 fold higher than IPTG induction levels, the significantly reduced cost of lactose ($7.00 per kilo, Sigma Chemical Co.) compared to IPTG ($20,000 perkilo, Sigma Chemical Co.) make the former inducer highly economically advantageous. For maximum production levels the metabolized substrate, lactose, must be replenished during cell growth or the culture will decrease the synthesis of the tac-promoted gene.

Materials and Methods

Cloning and DNA manipulation techniques are described in Maniatis, T., E. F. Fritsch and J. Sambrook (1982) Molecular Cloning, a Laboratory Manual. Cold Spring Harbor Laboratory Publishers, Cold Spring Harbor, N.Y.

A plasmid containing the lac operon, pSKS107, was received from Dr. M. Casadaban at the University of Chicago (Shapira, S. K., J. Chou, F. V. Richaud and M. J. Casadaban [1983] Gene 25:71-82). The sequence of the lacoperon is published (Kalnins, A., K. Otto, U. Ruther and B. Muller-Hill [1983] EMBO J. 2:593-597; Hediger, M. A., D. F. Johnson, D. P. Nierlich and I. Zabin [1985] Pro. Nat. Acad. Sci. 82:6414-6418). The lacI gene, on a 1.7 KB EcoRI restriction fragment cloned into pBR322, is available from the ATCC, as pMC9, catalogue no. 37195, 37196 and 37197. The sequence of the lacI gene is also published (Farabaugh, P. J. [1978] Nature 274:765-769). The broad host range vector, pMMB22, was received from Dr. M. Bagdasarian (Bagdasarian, M. M., E. Amann, R. Lurz, B. Ruckert and M. Bagdasarian [1983] Gene 26:273-282). A HindIII fragment of pMMB22, containing the lacI.sup.Q gene, was relinkered to BamHI. The sequence of the lacI.sup.Q 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 confirmed by DNA sequencing, e.g., the lacI and lacI.sup.Q promoters and the fusion region between the lacI transcriptional unit and the lac operon. The vector, pUC18, 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.DThesis, UCSD).

Following are examples which illustrate procedures, including the best mode, for practicing the invention. These examples should not be construedas limiting. All percentages are by weight and all solvent mixture proportions 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 first carried out in pUC18. Todo so, pUC18 was linearized with HindIII and then partially cut with HaeII.The DNA was resolved by electrophoresis on a 1.4% agarose gel. The 2402 bp band was eluted and ligated to a double-stranded synthetic DNA insert produced by .beta.-cyanoester chemistry on an Applied Biosystems 380A oligonucleotide synthesizer. This sequence starts at BP 1073 of the lacI sequence (Farabaugh, P. J., supra) as seen in Table 1.

__________________________________________________________________________ ##STR1## ##STR2## CC AAT ACG CAA ACC GCC TCT CCC CGC GCG TTG GCC GATCGCGGG TTA TGC GTT TGG CGG AGA GGG GCG CGC AAC CGG CTA ##STR3##TCA TTA ATG CAA CTC GCA CGA CAG GTC TCG AGA CTG GAA AGCAGT AAT TAC GTT GAG CGT GCT GTC CAG AGC TCT GAC CTT TCG ##STR4## S/D ##STR5## ##STR6##GGG CAG TGA GCGCTAGGAGGTAACTT ATG GAACCC GTC ACT CGCGATCCTCCATTGAA TAC CTTTCGA__________________________________________________________________________

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 lacI gene (BP 1137). These changes were introduced for ease in identification of the new construct. No amino acid changes would occur as a result of themutations introduced in making the base pair changes for the above two restriction sites. In this construct, the distance between the stop codon of lacI and the start codon of lacZ is 17 base pairs. This region containsa 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 inFIG. 1.

EXAMPLE 2

Confirmation 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 lacI gene of pMC9 to the lac operon found in pSKS107 (FIGS. 2a to d). After transformation, the lacI/Z fusion region was sequenced from a blue colony on an LB+X-gal (5-bromo-4-chloro-3-indolyl-.beta.-D-galactosidase, a colorimetric substrate for the presence of .beta.-galactosidase) plate. Those restriction sites that were examined yielded fragments of the predicted sized (FIGS. 2a to d). The lacI promoter region was also confirmed by sequence analysis.

EXAMPLE 3

Cloning

The EcoRI and SalI ends of the lacIZYA construct (FIGS. 2a to d) were respectively relinkered to yield BamHI and BglII restriction sites. The lacIZYA operon was cloned into the unique BamHI site of pMYC467 (FIG. 3) yielding pMYC471 (FIG. 4). The plasmid, pMYC467, contains the tac promotedlepidopteran toxin gene of pMYC436 (a cryIA(c)-like toxin gene [NRRL deposit no. B-18292] Adang, M. J. et. al. [1985] Gene 36:289-300) cloned into the broad host range vector, pTJS260. pMYC471 was introduced by transformation into P. flourescens MB101 and the resulting clone was designated MR471. MR471 was tested for the key elements of the resident pMYC471 plasmid. A functional repressor is synthesized because in the absence of IPTG or lactose no significant amount of toxin is produced (Table 1). The cells are blue when plated on X-gal because a functional .beta.-galactosidase is present. A functional permease and .beta.-galactosidase are present because the cells are able to induce the tac promoter in the presence of lactose (Table 1).

The subject invention can be used with any heterologous gene to control itsexpression from lac operated promoters. The expression product (protein) can be isolated from the culture medium of the producing microbe by means known in the art for isolating such a product from microbial cultures. Alternatively, a product which remains intracellular can be used in the form of the microbe itself, for example, as a biological insecticide. See U.S. Pat. Nos. 4,695,455 and 4,695,462 for such uses.

TABLE 1__________________________________________________________________________ 1 GACACCATCG ##STR7## GGTATGGCAT GATAGCGCCC 51 GGAAGAGAGT ##STR8## GAAACCAGTA ACGTTATACG 101 ATGTCGCAGA GTATGCCGGT GTCTCTTATC AGACCGTTTC CCGCGTGGTG 151 AACCAGGCCA GCCACGTTTC TGCGAAAACG CGGGAAAAAG TGGAAGCGGC 201 GATGGCGGAG CTGAATTACA TTCCCAACCG CGTGGCACAA CAACTGGCGG 251 GCAAACAGTC GTTGCTGATT GGCGTTGCCA CCTCCAGTCT GGCCCTGCAC 301 GCGCCGTCGC AAATTGTCGC GGCGATTAAA TCTCGCGCCG ATCAACTGGG 351 TGCCAGCGTG GTGGTGTCGA TGGTAGAACG AAGCGGCGTC GAAGCCTGTA 401 AAGCGGCGGT GCACAATCTT CTCGCGCAAC GCGTCAGTGG GCTGATCATT 451 AACTATCCGC TGGATGACCA GGATGCCATT GCTGTGGAAG CTGCCTGCAC 501 TAATGTTCCG GCGTTATTTC TTGATGTCTC TGACCAGACA CCCATCAACA 551 GTATTATTTT CTCCCATGAA GACGGTACGC GACTGGGCGT GGAGCATCTG 601 GTCGCATTGG GTCACCAGCA AATCGCGCTG TTAGCGGGCC CATTAAGTTC 651 TGTCTCGGCG CGTCTGCGTC TGGCTGGCTG GCATAAATAT CTCACTCGCA 701 ATCAAATTCA GCCGATAGCG GAACGGGAAG GCGACTGGAG TGCCATGTCC 751 GGTTTTCAAC AAACCATGCA AATGCTGAAT GAGGGCATCG TTCCCACTGC 801 GATGCTGGTT GCCAACGATC AGATGGCGCT GGGCGCAATG CGCGCCATTA 851 CCGAGTCCGG GCTGCGCGTT GGTGCGGATA TCTCGGTAGT GGGATACGAC 901 GATACCGAAG ACAGCTCATG TTATATCCCG CCGTCAACCA CCATCAAACA 951 GGATTTTCGC CTGCTGGGGC AAACCAGCGT GGACCGCTTG CTGCAACTCT1001 CTCAGGGCCA GGCGGTGAAG GGCAATCAGC TGTTGCCCGT CTCACTGGTG1051 AAAAGAAAAA CCACCCTGGC GC'CCAATACG CAAACCGCCT CTCCCCGCGC1101 GTTGGCCGAT TCATTAATGC AACTCGCACG ACAGGTCTCG AGACTGGAAA 1151 ##STR9##1201 TCGTTTTACA ACGTCGTGAC TGGGAAAACC CTGGCGTTAC CCAACTTAAT1251 CGCCTTGCAG CACATCCCCC TTTCGCCAGC TGGCGTAATA GCGAAGAGGC1301 CCGCACCGAT CGCCCTTCCC AACAGTTGCG CAGCCTGAAT GGCGAATGGC1351 GCTTTGCCTG GTTTCCGGCA CCAGAAGCGG TGCCGGAAAG CTGGCTGGAG1401 TGCGATCTTC CTGAGGCCGA TACTGTCGTC GTCCCCTCAA ACTGGCAGAT1451 GCACGGTTAC GATGCGCCCA TCTACACCAA CGTAACCTAT CCCATTACGG1501 TCAATCCGCC GTTTGTTCCC ACGGAGAATC CGACGGGTTG TTACTCGCTC1551 ACATTTAATG TTGATGAAAG CTGGCTACAG GAAGGCCAGA CGCGAATTAT1601 TTTTGATGGC GTTAACTCGG CGTTTCATCT GTGGTGCAAC GGGCGCTGGG1651 TCGGTTACGG CCAGGACAGT CGTTTGCCGT CTGAATTTGA CCTGAGCGCA1701 TTTTTACGCG CCGGAGAAAA CCGCCTCGCG GTGATGGTGC TGCGTTGGAG1751 TGACGGCAGT TATCTGGAAG ATCAGGATAT GTGGCGGATG AGCGGCATTT1801 TCCGTGACGT CTCGTTGCTG CATAAACCGA CTACACAAAT CAGCGATTTC1851 CATGTTGCCA CTCGCTTTAA TGATGATTTC AGCCGCGCTG TACTGGAGGC1901 TGAAGTTCAG ATGTGCGGCG AGTTGCGTGA CTACCTACGG GTAACAGTTT1951 CTTTATGGCA GGGTGAAACG CAGGTCGCCA GCGGCACCGC GCCTTTCGGC2001 GGTGAAATTA TCGATGAGCG TGGTGGTTAT GCCGATCGCG TCACACTACG2051 TCTGAACGTC GAAAACCCGA AACTGTGGAG CGCCGAAATC CCGAATCTCT2101 ATCGTGCGGT GGTTGAACTG CACACCGCCG ACGGCACGCT GATTGAAGCA2151 GAAGCCTGCG ATGTCGGTTT CCGCGAGGTG CGGATTGAAA ATGGTCTGCT2201 GCTGCTGAAC GGCAAGCCGT TGCTGATTCG AGGCGTTAAC CGTCACGAGC2251 ATCATCCTCT GCATGGTCAG GTCATGGATG AGCAGACGAT GGTGCAGGAT2301 ATCCTGCTGA TGAAGCAGAA CAACTTTAAC GCCGTGCGCT GTTCGCATTA2351 TCCGAACCAT CCGCTGTGGT ACACGCTGTG CGACCGCTAC GGCCTGTATG2401 TGGTGGATGA AGCCAATATT GAAACCCACG GCATGGTGCC AATGAATCGT2451 CTGACCGATG ATCCGCGCTG GCTACCGGCG ATGAGCGAAC GCGTAACGCG2501 AATGGTGCAG CGCGATCGTA ATCACCCGAG TGTGATCATC TGGTCGCTGG2551 GGAATGAATC AGGCCACGGC GCTAATCACG ACGCGCTGTA TCGCTGGATC2601 AAATCTGTCG ATCCTTCCCG CCCGGTGCAG TATGAAGGCG GCGGAGCCGA2651 CACCACGGCC ACCGATATTA TTTGCCCGAT GTACGCGCGC GTGGATGAAG2701 ACCAGCCCTT CCCGGCTGTG CCGAAATGGT CCATCAAAAA ATGGCTTTCG2751 CTACCTGGAG AGACGCGCCC GCTGATCCTT TGCGAATACG CCCACGCGAT2801 GGGTAACAGT CTTGGCGGTT TCGCTAAATA CTGGCAGGCG TTTCGTCAGT2851 ATCCCCGTTT ACAGGGCGGC TTCGTCTGGG ACTGGGTGGA TCAGTCGCTG2901 ATTAAATATG ATGAAAACGG CAACCCGTGG TCGGCTTACG GCGGTGATTT2951 TGGCGATACG CCGAACGATC GCCAGTTCTG TATGAACGGT CTGGTCTTTG3001 CCGACCGCAC GCCGCATCCA GCGCTGACGG AAGCAAAACA CCAGCAGCAG3051 TTTTTCCAGT TCCGTTTATC CGGGCAAACC ATCGAAGTGA CCAGCGAATA3101 CCTGTTCCGT CATAGCGATA ACGAGCTCCT GCACTGGATG GTGGCGCTGG3151 ATGGTAAGCC GCTGGCAAGC GGTGAAGTGC CTCTGGATGT CGCTCCACAA3201 GGTAAACAGT TGATTGAACT GCCTGAACTA CCGCAGCCGG AGAGCGCCGG3251 GCAACTCTGG CTCACAGTAC GCGTAGTGCA ACCGAACGCG ACCGCATGGT3301 CAGAAGCCGG GCACATCAGC GCCTGGCAGC AGTGGCGTCT GGCGGAAAAC3351 CTCAGTGTGA CGCTCCCCGC CGCGTCCCAC GCCATCCCGC ATCTGACCAC3401 CAGCGAAATG GATTTTTGCA TCGAGCTGGG TAATAAGCGT TGGCAATTTA3451 ACCGCCAGTC AGGCTTTCTT TCACAGATGT GGATTGGCGA TAAAAAACAA3501 CTGCTGACGC CGCTGCGCGA TCAGTTCACC CGTGCACCGC TGGATAACGA3551 CATTGGCGTA AGTGAAGCGA CCCGCATTGA CCCTAACGCC TGGGTCGAAC3601 GCTGGAAGGC GGCGGGCCAT TACCAGGCCG AAGCAGCGTT GTTGCAGTGC3651 ACGGCAGATA CACTTGCTGA TGCGGTGCTG ATTACGACCG CTCACGCGTG3701 GCAGCATCAG GGGAAAACCT TATTTATCAG CCGGAAAACC TACCGGATTG3751 ATGGTAGTGG TCAAATGGCG ATTACCGTTG ATGTTGAAGT GGCGAGCGAT3801 ACACCGCATC CGGCGCGGAT TGGCCTGAAC TGCCAGCTGG CGCAGGTAGC3851 AGAGCGGGTA AACTGGCTCG GATTAGGGCC GCAAGAAAAC TATCCCGACC3901 GCCTTACTGC CGCCTGTTTT GACCGCTGGG ATCTGCCATT GTCAGACATG3951 TATACCCCGT ACGTCTTCCC GAGCGAAAAC GGTCTGCGCT GCGGGACGCG4001 CGAATTGAAT TATGGCCCAC ACCAGTGGCG CGGCGACTTC CAGTTCAACA4051 TCAGCCGCTA CAGTCAACAG CAACTGATGG AAACCAGCCA TCGCCATCTG4101 CTGCACGCGG AAGAAGGCAC ATGGCTGAAT ATCGACGGTT TCCATATGGG 4151 GATTGGTGGC GACGACTCCT GGAGCCCGTC AGTATCGGCG ##STR10## 4201 TGAGCGCCGG TCGCTACCAT TACCAGTTGG TCTGGTGTCA ##STR11## 4251 TAACCGGGCA GGCCATGTCT GCCCGTATTT CGCGTAAGGA ##STR12##4301 --GTACTATTTA AAAAACACAA ACTTTTGGAT GTTCGGTTTA TTCTTTTTCT4351 TTTACTTTTT TATCATGGGA GCCTACTTCC CGTTTTTCCC GATTTGGCTA4401 CATGACATCA ACCATATCAG CAAAAGTGAT ACGGGTATTA TTTTTGCCGC4451 TATTTCTCTG TTCTCGCTAT TATTCCAACC GCTGTTTGGT CTGCTTTCTG4501 ACAAACTCGG GCTGCGCAAA TACCTGCTGT GGATTATTAC CGGCATGTTA4551 GTGATGTTTG CGCCGTTCTT TATTTTTATC TTCGGGCCAC TGTTACAATA4601 CAACATTTTA GTAGGATCGA TTGTTGGTGG TATTTATCTA GGCTTTTGTT4651 TTAACGCCGG TGCGCCAGCA GTAGAGGCAT TTATTGAGAA AGTCAGCCGT4701 CGCAGTAATT TCGAATTTGG TCGCGCGCGG ATGTTTGGCT GTGTTGGCTG4751 GGCGCTGTGT GCCTCGATTG TCGGCATCAT GTTCACCATC AATAATCAGT4801 TTGTTTTCTG GCTGGGCTCT GGCTGTGCAC TCATCCTCGC CGTTTTACTC4851 TTTTTCGCCA AAACGGATGC GCCCTCTTCT GCCACGGTTG CCAATGCGGT4901 AGGTGCCAAC CATTCGGCAT TTAGCCTTAA GCTGGCACTG GAACTGTTCA4951 GACAGCCAAA ACTGTGGTTT TTGTCACTGT ATGTTATTGG CGTTTCCTGC5001 ACCTACGATG TTTTTGACCA ACAGTTTGCT AATTTCTTTA CTTCGTTCTT5051 TGCTACCGGT GAACAGGGTA CGCGGGTATT TGGCTACGTA ACGACAATGG5101 GCGAATTACT TAACGCCTCG ATTATGTTCT TTGCGCCACT GATCATTAAT5151 CGCATCGGTG GGAAAAACGC CCTGCTGCTG GCTGGCACTA TTATGTCTGT5201 ACGTATTATT GGCTCATCGT TCGCCACCTC AGCGCTGGAA GTGGTTATTC5251 TGAAAACGCT GCATATGTTT GAAGTACCGT TCCTGCTGGT GGGCTGCTTT5301 AAATATATTA CCAGCCAGTT TGAAGTGCGT TTTTCAGCGA CGATTTATCT5351 GGTCTGTTTC TGCTTCTTTA AGCAACTGGC GATGATTTTT ATGTCTGTAC5401 TGGCGGGCAA TATGTATGAA AGCATCGGTT TCCAGGGCGC TTATCTGGTG5451 CTGGGTCTGG TGGCGCTGGG CTTCACCTTA ATTTCCGTGT TCACGCTTAG 5501 CGGCCCCGGC CCGCTTTCCC TGCTGCGTCG TCAGGTGAAT ##STR13##5551 .sub.----AAGCAATCAA TGTCGGATGC GGCGCGACGC TTATCCGACC AACATATCAT 5601 AACGGAGTGA ##STR14## ACCGAAAGAA TAAGAGCAGG5651 CAAGCTATTT ACCGATATGT GCGAAGGCTT ACCGGAAAAA AGACTTCGTG5701 GGAAAACGTT AATGTATGAG TTTAATCACT CGCATCCATC AGAAGTTGAA5751 AAAAGAGAAA GCCTGATTAA AGAAATGTTT GCCACGGTAG GGGAAAACGC5801 CTGGGTAGAA CCGCCTGTCT ATTTCTCTTA CGGTTCCAAC ATCCATATAG5851 GCCGCAATTT TTATGCAAAT TTCAATTTAA CCATTGTCGA TGACTACACG5901 GTAACAATCG GTGATAACGT ACTGATTGCA CCCAACGTTA CTCTTTCCGT5951 TACGGGACAC CCTGTACACC ATGAATTGAG AAAAAACGGC GAGATGTACT6001 CTTTTCCGAT AACGATTGGC AATAACGTCT GGATCGGAAG TCATGTGGTT6051 ATTAATCCAG GCGTCACCAT CGGGGATAAT TCTGTTATTG GCGCGGGTAG6101 TATCGTCACA AAAGACATTC CACCAAACGT CGTGGCGGCT GGCGTTCCTT6151 GTCGGGTTAT TCGCGAAATA AACGACCGGG ATAAGCACTA TTATTTCAAA 6201 GATTATAAAG TTGAATCGTC ##STR15## CCTGATACGC6251 TGCGCTTATC AGGCCTACAA GTTCAGCGAT CTACATTAGC CGCATCCGGC6301 ATGAACAAAG CGCAGGAACA AGCGTCGCAT CATGCCTCTT TGACCCACAG6351 CTGCGGAAAA CGTACTGGTG CAAAACGCAG GGTTATGATC ATCAGCCCAA6401 CGACGCACAG CGCATGAAAT GCCCAGTCCA TCAGGTAATT GCCGCTGATA6451 CTACGCAGCA CGCCAGAAAA CCACGGGGCA AGCCCGGCGA TGATAAAACC6501 GATTCCCTGC ATAAACGCCA CCAGCTTGCC AGCAATAGCC GGTTGCACAG6551 AGTGATCGAG CGCCAGCAGC AAACAGAGCG GAAACGCGCC GCCCAGACCT6601 AACCCACACA CCATCGCCCA CAATACCGGC AATTGCATCG GCAGCCAGAT6651 AAAGCCGCAG AACCCCACCA GTTGTAACAC CAGCGCCAGC ATTAACAGTT6701 TGCGCCGATC CTGATGGCGA GCCATAGCAG GCATCAGCAA AGCTCCTGCG6751 GCTTGCCCAA GCGTCATCAA TGCCAGTAAG GAACCGCTGT ACTGCGCGCT6801 GGCACCAATC TCAATATAGA AAGCGGGTAA CCAGGCAATC AGGCTGGCGT6851 AACCGCCGTT AATCAGACCG AAGTAAACAC CCAGCGTCCA CGCGCGGGGA6901 GTGAATACCA CGCGAACCGG AGTGGTTGTT GTCTTGTGGG AAGAGGCGAC6951 CTCGCGGGCG CTTTGCCACC ACCAGGCAAA GAGCGCAACA ACGGCAGGCA7001 GCGCCACCAG GCGAGTGTTT GATACCAGGT TTCGCTATGT TGAACTAACC7051 AGGGCGTTAT GGCGGCACCA AGCCCACCGC CGCCCATCAG AGCCGCGGAC7101 CACAGCCCCA TCACCAGTGG CGTGCGCTGC TGAAACCGCC GTTTAATCAC7151 CGAAGCATCA CCGCCTGAAT GATGCCGATC CCCACCCCAC CAAGCAGTGC7201 GCTGCTAAGC AGCAGCGCAC TTTGCGGGTA AAGCTCACGC ATCAATGCAC7251 CGACGGCAAT CAGCAACAGA CTGATGGCGA CACTGCGACG TTCGCTGACA7301 TGCTGATGAA GCCAGCTTCC GGCCAGCGCC AGCCCGCCCA TGGTAACCAC 7351 CGGCAGAGCG ##STR16##__________________________________________________________________________(1) sequence in bold, between the two ' marks, is synthetic DNA used to fuse the lac operon to the lac I or lac I.sup.Q gene operon.(2) pSKS107 contains an unsequenced mutation at the EcoRI site normally found in the lac Z gene. TABLE 2______________________________________Lactose Inducibility of Various ConstructsHours after.sup.(1) Lactose Toxin.sup.(2)Induction (mM) Cells/ml (ug/ml)______________________________________ MR47140 0 1.6 .times. 10.sup.10 none detected40 no lactose 2 mM IPTG 2.3 .times. 10.sup.10 100315 20 9.1 .times. 10.sup.9 101124 20 1.2 .times. 10.sup.10 73715 40 1.8 .times. 10.sup.10 88724 40 1.1 .times. 10.sup.10 102523.sup.(3) 8.3.sup.(4) 1.9 .times. 10.sup.10 93839.sup.(3) 8.3.sup.(4) 2.0 .times. 10.sup.10 1555 MB101 containing pMYC48524 40 -- 77524 0 -- none detected MB101 containing pMYC116124 40 -- 30024 0 -- none detected______________________________________ .sup.(1) Cultures were induced upon reaching stationary phase. .sup.(2) Toxin concentration was determined by laser densitometry of Coomassiestained protein bands after electrophoresis of disrupted cells oSDSPAGE (LKB Instructional Manual 222010). .sup.(3) This experiment used MR471 grown in a 10L fermentor. All other experimental data were generated using same medium in 250 ml baffled shakflasks. .sup.(4) In the fermentor, 8.3 mM lactose/hour was fed into the culture. It was found that MR471 did not metabolize this lactose level in a fermentor, resulting in increased concentrations during the experiment. The experiments done in shake flasks were given a single dose of lactose or IPTG at the indicated times.

Claims

1. A novel lacI ZYA operon having the sequence shown in Table 1, or mutations thereof which retain the ability to control expression of heterologous genes from lac operated promoters as does the parent operon.

2. A novel lacI.sup.Q ZYA operon having the sequence shown in Table 1, wherein base 16 is a T instead of a C as shown in the table, or mutations thereof which retain the ability to control expression of heterologous genes from lac operated promoters as does the parent operon.

3. A process for controlling the expression of a heterologous gene from a lac operated promoter which comprises culturing a microbe comprising a heterologous gene expressed from a lac operated promoter, and a lacIZYA or lacI.sup.Q ZYA operon wherein the CAP binding site and lac promoter/operator is not present in the lac operon.

4. The process, according to claim 3, wherein said microbe has been transformed with a plasmid comprising the heterologous gene.

5. The process according to claim 3, wherein said microbe comprises a chromosome comprising a lacIZYA or lacI.sup.Q ZYA operon wherein the CAP binding site and lac promoter/operator is not present in the lac operon.

6. A transfer vector comprising a lacIZYA or lacI.sup.Q 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 vector of claim 6.

8. The transfer vector, according to claim 6, being a plasmid.

9. A process for preparing a protein product which comprises culturing a microbe comprising a heterologous gene encoding said product which is expressed from a lac promoter, and a lacIZYA or lacI.sup.Q ZYA operon wherein the CAP binding site and lac promoter/operator is not present in the lac operon, until a sufficient amount of desired product is produced.

10. A process for controlling the expression of a heterologous gene from a lac operated promoter which comprises culturing a microbe comprising a heterologous gene expressed from a lac operated promoter, and a lacIZY or lacI.sup.Q ZY operon wherein the CAP binding site and lac promoter/operator is not present in the lac operon.

11. The process, according to claim 10, wherein said microbe has been transformed with a plasmid comprising a heterologous gene.

12. The process according to claim 10, wherein said microbe comprises a chromosome comprising said lacIZY or lacI.sup.Q ZY operon wherein the CAP binding site and lac promoter/operator is not present in the lac operon.

13. A transfer vector comprising a lacIZY or lacI.sup.Q ZY 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 vector of claim 13.

15. The transfer vector, according to claim 13, being a plasmid.

16. A process for preparing a protein product which comprises culturing a microbe comprising a heterologous gene encoding said product which is expressed from a lac promoter, and a lacIZY or lacI.sup.Q ZY operon wherein the CAP binding site and lac promoter/operator is not present in the lac operon, until a sufficient amount of desired product is produced.