RU2124559C1 - RECOMBINANT PLASMID DNA, METHOD OF EXPRESSION OF α-AMYLASE GENE IN STREPTOMYCES CELLS - Google Patents

Abstract

FIELD: biotechnology, molecular biology, genetic engineering. SUBSTANCE: recombinant plasmid DNA pULDVI consists of vector plasmid pIJ-699 and DNA sequence. DNA sequence is inserted into Bgl II-site of plasmid pIJ-699 and has SAF-promoter and a sequence operably bound with 5'-end of complete sequence of amy-gene located in Hind III-fragment (size is 4.8 kb) from genome DNA of S. griseus. Plasmid DNA has unique sites: for Hind III, Nco I, Bst EII, Sal I and Bgl II. Invention describes an isolation of complete sequence of gene encoding α-amylase containing promoter, site encoding signal peptide and a sequence encoding an enzyme protein. The indicated gene sequence is bound with SAF-promoter and inserted into vector followed by transformation with obtained recombinant vector of Streptomyces strain. Transformants are selected, cultured their under conditions providing expression of the inserted gene. EFFECT: improved expression of foreign DNA sequence in Streptomyces cells. 3 cl, 3 tbl, 15 dwg

Description

 The invention relates to the field of biotechnology. The present invention relates to a Streptomyces expression system and a method for expressing foreign DNA sequences in Streptomyces, as well as providing secretion of polypeptides and proteins encoded by a foreign DNA sequence.

 Species of Streptomyces are well-known producers of a variety of extracellular enzymes, including proteases, phosphatases, xylanases, celluloses, amylases, lipases, and nucleases.

 In addition, members of the Streptomyces genus produce a large number of antibiotics, pigments, and other secondary metabolites and have properties that require a complex differentiation technique that leads to spore formation. In experimental Streptomyces culture series, there is usually a match in the production of extracellular enzymes, the activation of antibiotic production, pigment biosynthesis and sporulation. All these processes are suppressed by nutritional conditions favoring a high growth rate and are activated by limiting the sources of phosphorus, or carbon, or nitrogen. It is likely that the secretion of enzymes, the formation of secondary metabolites and differentiation are completely independent, but respond to similar triggers.

 Previously, some Streptomyces genes encoding extracellular enzymes have been cloned. These enzymes include Streptomyces coelicolor agarose, Streptomyces plicatus endoglycosidase H, Streptomyces Lividans xylanase, Streptomyces hydroscopicus alpha amylase, Streptomyces SpA2 strain, strep beta-galactosidase. Lividans and beta-lactamases from strep. cocaoi badius and fradial.

 However, the regulatory mechanisms that control the expression of these genes are still unknown. In addition to specific regulatory mechanisms, such as the induction of amylase by dextrins or maltotriose and the carbon-metabolite regulation of amylase or agarase, the suppression of the formation of several extracellular enzymes is likely to be removed, since after the decrease in nutrition, streptomyces simultaneously produced several enzymes that degrade polymer substrates. Such transactivating regulatory genes were found in Bacillus subtilis (J. Bacterid 169: 324 - 333, 1987), Bacillus natto (J. Bacteriol. 166: 20 - 28, 1986) and Bacillus Licheniformis.

 Positive regulatory genes that affect enzyme synthesis and / or secretion could be cloned when looking for increased secretion of extracellular enzymes in weakly secreting strains like S. Lividans.

 Systems for the expression of foreign DNA sequences in streptomyces have previously been described, for example, in EP 148.552 and WO-88/07079. These systems utilize the endogenous promoters of extracellular enzymes produced by streptomyces.

 The main object of the invention is a method for producing extracellular enzymes or a desired polypeptide by culturing a transformed host organism according to the present invention and isolating the product from the culture broth.

 Another aspect of the invention is the saf gene promoter (i.e., a gene encoding a secondary metabolism activating factor).

 And another aspect of the invention is the production of cloning carriers (vectors) that include a saf gene promoter operably linked to the signaling region of endogenous extracellular enzyme secretion, and both are operably linked to a foreign DNA sequence; this aspect also relates to organisms or host cells transformed with such cloning carriers, resulting in the expression and secretion of a foreign polypeptide encoded by a foreign DNA sequence.

 Another aspect of the invention is the integration of such a cloning carrier carrying a foreign DNA sequence into the streptomyces chromosomal DNA. In a preferred embodiment, an expression vector containing a saf gene is also incorporated into such transformable organisms.

 These and other objects of the invention are described in more detail in the following description.

 A brief description of the invention.

 A DNA fragment from the ATCC strain 10137 streptomyces qriseus encoding the saf gene (secondary metabolism activation factor) gene, which is included in the usual mechanism for controlling at least five enzymes secreted by cells of the species S. Lividans and S. coelicolor. This saf gene is present in all streptomyces tested, suggesting an important role that the gene plays in metabolism. Indeed, a DNA fragment containing a gene similar to the saf gene was cloned by the applicant from streptomyces oriseus IMPV 3570, a candidicidin producing strain. In some streptomyces, for example, S. Lividans and S. lactamdurans, a hybridization model suggests that the saf gene is present in several copies of the chromosomal DNA.

 The following is a first example of a gene involved in the production of extracellular enzymes and sporulation in streptomyces.

 The saf gene encodes a 113 amino acid polypeptide (SAF polypeptide); an in vitro transcription-translation study in E. coli showed that a protein of the correct size is formed, about 15,000 daltons, the SAF polypeptide has a strong positive charge (18 positively charged amino acids) and does not show the composition characteristic of a leader peptide or transmembrane protein and therefore represents direct effect on protein excretion is unlikely. A positively charged protein can easily interact with DNA. Indeed, a DNA-binding region typical of several regulatory proteins is observed in the SAF polypeptide (Ann. Rew. Biochem. 53: 293 - 321, 1984). It has been unexpectedly discovered that the saf promoter is more efficient than the natural promoter of extracellular enzymes. For example, the amylase gene produces more amylase when the saf promoter replaces the natural amylase promoter. Thus, the saf promoter can be used to increase the expression of any endogenous polypeptide or protein instead of the natural protein promoter.

 The SAF polypeptide has a pleiotropic effect, i.e., has an effect in more than one direction on extracellular enzymes, on pigment production and on differentiation. In accordance with the invention, increased production of endogenous (extracellular) proteins is achieved.

 More generally, the saf gene and the corresponding polypeptide can be used to increase the expression of selected heterologous proteins in streptomyces by inserting the gene encoding the desired protein into a suitable cleavage site of the extracellular enzyme encoding SAF, or parts thereof, transforming the streptomyces host containing saf gene with a recombined gene, and culturing the transformed bacteria to isolate the selected protein or part thereof. Preferably, heterologous DNA, under the control of the saf promoter, is inserted (integrated) into the chromosomal DNA, and an expression vector containing DNA encoding the SAF polypeptide is inserted with it. The expression vector does not need to be integrated into chromosomal DNA.

 Pleiotropic genes affecting antibiotic biosynthesis and differentiation in streptomyces have been reported. The afSB gene, which positively controls the production of A-factor, actinorodine, and prodigiosin in streptomyces coelicolor and streptomyces lividans, was cloned from s. coelicolor A3- / 2- / (Horinouchi et al., J. Bacteriol. 155: 1238 - 1248, 1983).

 The saf gene is different from afSB and, indeed, they have the opposite effect on the production of active rhodin. What they have in common is that their amino acid sequences are typical of DNA-binding proteins. Both saf and afSB genes have promoters without -10 and -35 matching sequences and are linked by potential translational regulatory signals (factors), and a strong trunk, and loop structures that can act as transcriptional terminators (Horinouchi et al., J. Bacteriol. 168: 257-269, 1986). If transcription forms a very stable loop, the binding of ribosomes will become impossible. Such "weakening" mechanisms of control of gene expression have been found for the controlled control of antibiotic resistance genes in streptomyces (Horinouchi et al., Proc. Natl. Acad. Sci. USA, 77: 7079 - 7083, 1989). Since the saf gene can be thought of as the “main” gene, which includes a variety of genes for the excretion of enzymes, its expression is probably strictly controlled in the cell. All enzymes stimulated by the saf gene are involved in the decomposition of complex polymers and therefore we believe that this gene is part of the general regulatory system involved in the search for alternative food sources, as is also suggested by Bacillus (Henner et al., J. Bacteriol 170: 296-300, 1988).

 FIG. 1 - Maps of the restriction of fragments of chromosomal DNA from s. grisous ATCC 10137 clones of Bgl II site pIJ 702 that carry the genetic determinant of superproduction of alkaline phosphatase.

 FIG. 2 - Analysis (southern hybridization) of homology between 1 kb Bgl II fragment pIJ AD3 and the genomic DNA of other streptomyces species.

 A / DNA fragments obtained by Ba mHI by digestion of genomic DNA (lanes 2-7).

 B / DNA hybridization shown in panel A, with a 1 kb fragment as a sample / probe /.

Bands: 1. DNA digested with Hind III to obtain fragments of sizes 23, 9.59, 6.68, 4.29, 2.28, 1.94 and 0.58 kb;
2. S. lactamdurans;
3. S. acrimycini;
4. S. coelicolor 1157;
5. S. qriseus 2212;
6. S. qriseus 3570;
7. S. Lividans 1326.

 FIG. 3 Experiments on testing the protease / A /, amylase / B / and lipase / C / activity of S. Lividans transformed with pIJ 702 / left / and transformed with p VL AD3 / right /.

 FIG. 4 Effect of gene dosage on saf gene expression. The production of protease / A / and amylase / B / S. Lividans transformed with pIJ 702 / left / S. Lividans transformed with p VLAD3 / in the middle / and S. Lividans transformed with p VLAD3 / right /.

 FIG. 5 Trimming / "trimming" / saf-gene. 1 kb Bgl II. Fragment p VLAD3 was used as starting material. Extracellular enzyme production for all plasmid constructs was measured on plates with solid medium, as described, means wild type producer, two, three or four crosses indicate different degrees of superproduction. Short open arrows indicate the localization and direction of transcription of the tyrosinase gene / mel / in plasmid pIJ 702. Closed circles indicate the mel gene promoter; open / bright / circles represent the activity of the putative clockwise promoter in pIJ 702, and open squares show the saf promoter. The direction of transcription is indicated by arrows, ORFI corresponds to the saf reading frame, derived from the nucleotide sequence data (see FIG. 6A).

 FIG. 6 The nucleotide sequence of 1 kb. Bgl II fragment p of VLAD3 and FIG. 6A is the deduced amino acid sequence of the saf gene product. The second codon of the putative ATG initiation is underlined. Inverted complementary repeat sequences are shown by converging arrows. The wavy line shows an amino acid sequence similar to the DNA-binding regions of known DNA-binding proteins. Suitable restriction sites are shown. Nucleotides are numbered starting from the Bgl II site / number on the right /.

 FIG. 7 Comparison of the region from the deduced amino acid sequence of the saf gene product with DNA binding regions in known DNA binding proteins. Data taken from Pabo and Sauer, Ann. Rev. Biochem. 53: 293 - 321, 1984 and Horinouchi and Beppu, Genetics of Industrial Microorganisms p. 41, ed. Pliva, Zagreb, Yugoslavia, 1986.

 FIG. 8 Repeated up and down nucleotide sequences from the saf gene.

 FIG. 9 Repeated nucleotide groups present in the saf gene.

 FIG. 10 Plasmid pIJ 702.

 In FIG. 11 shows the complete nucleotide sequence of the deduced amino acid sequence of the amylase gene of S. griseus.

 It is believed that the Bst EII incision is the optimal site for cross-linking a foreign gene.

 FIG. 12 Strategy used for cloning the a-amylase gene of S. griseus strain IMRU 3570 using plasmid pIJ 699.

p ULVD 10 в ММ 1 мМР + 1% крахмала + тио.FIG. 13 A series of graphs showing the production of a-amylase on plates with different plasmids versus time, with different media composition:
(Δ) MM + starch / 1% / + thio
(□) MM 1 mMR + starch / 1% / + thio
(■) MM without phosphate + starch / 1% / + thio
(•) MM + starch / 1% / + thio + IRTG
The lower panel on the right side shows the estimates of the production of the plasmid PUL VD10 compared to pULTVI, S. Lividans and p ULTV 100 in the above environments in the best production medium (Fig. 6a) S. Lividans (pIJ 699) in MM without phosphate +% starch + tio: / ^. / p ULTVI in the above environments; / - / p ULTV 100 in the above environments; and

p ULVD 10 in MM 1 mMR + 1% starch + thio.

 FIG. 14. Schematically represents the construction of a recombinant streptomyces cell containing a synthesis protein producing chromosome gene including a CRF molecule and plasmids containing DNA: an SAF polypeptide encoding a polypeptide.

 The work described here was carried out using the following materials and methods.

 Bacterial strains and plasmids. In this study, streptomyces and E. coli strains were used, which are shown in Table 1. Plasmids and phages are shown in table. 2.

Cultivation media and conditions. Streptomyces strains were grown in P2UE, minimal medium / MM /, TSB / Difco / or YEME supplemented with 34% sucrose and 5 mM MgCl ₂ / Hopwood et al., Genetic Manipulation of streptomyces, Laboratory Textbook, ed. John Innes Foundation, Norwich, UK, 1985 /. Strains of streptomyces were grown in triple blocked flasks at 28 ^o C in a rotary shaker during its rotation at a speed of 220 rpm.

Strains of E. coli were grown in Luria broth / LB / (see Miller et al., EXPERIMENTS in Molecular genetics p. 433, Cold Spring Harbor Lab. New York 1972) or Luria agar / LA / at 37 ^o C.

 Cup experiments with enzymes. The alkaline phosphatase streptomyces assay was carried out in PPMM / MM / without glucose containing a reduced level of / 1 mM / phosphatase supplemented with 30 μg / ml 5-bromo-4-chloro-3-indolylphosphate-r-toluidine / XP /. The colonies producing alkaline phosphatase were blue, and those unable to produce it were white. For E. coli, BXR medium was used, which contains (per liter): 10 g bactopecton, 12 g Trisma base, 10 g salt, 20 g Difco agar, with a pH of 7.5 and 40 μg / ml XP /.

Amylase activity of streptomyces colonies was tested on MM (glucose-free) with 1% starch. After 3 days of growth, the plates were treated with iodine vapor. Zones of enlightenment around the colonies result from the decomposition of starch. Lipase activity was measured by growing streptomyces in MM supplemented with 2% / weight / volume / olive oil, 0.5 / weight / volume / tween 80 and 0.5% / weight / volume / tween 20. After 20 days of growth, 1 ml of 1M CaCl ₂ was poured into cups.

Lipase production was observed as a precipitate of Ca ²⁺ -fatty acid complex / Odera et al., Ferment. Technol. 64: 363 - 371, 1986 /.

Proteasun activity was tested in MM / without glucose with the addition of 0.5% casein and 10 mm CaCl ₂ . Enzyme activity was defined as clarification zones around colonies.

 Agarose activity of S. coelicolor was tested in glucose-free MM with cup filling with a gram-iodine solution. Beta-galactisidase was observed as blue staining of colonies growing on MM without glucose with the addition of X-gal / 36 μg / ml / and IRTG / 10 μg / ml /.

 DNA isolation. Total / all / DNA from streptomyces was prepared as described by Hopwood et al. Laboratory Textbook (see above). Plasmid DNA from streptomyces or E. coli was isolated according to the method of Kizer / Kieser et al., Plasmid 12: 19–36, 1984 /.

Cloning Procedures 10 μg of the chromosomal DNA of S. griseus ATCC 10137 strain and 0.5 μg pIJ 702 were completely digested with Bgl II and ligated for 12 hours at 14 ^° C using T4 DNA ligase. Ligation mixture was used directly for transformation. The DNA fragments were subcloned by digesting 1-2 μg of plasmid DNA with an adequate restriction enzyme (s), and the reaction products were separated by gel electrophoresis in low melting point agarose (LMPA). The required DNA strands were extracted using methods with CTAB / Langridge et al., Anal. Biochem. 103: 264-272, 1980 /.

Methods of transformation. The streptomyces strains were transformed as described by Hopwood et al. In the Laboratory Textbook (see above). After transformation, protoplasts were placed on R2YE medium and left to generate for 15-20 hours at 30 ^° C. Then, 1 ml of an aqueous solution of thiostrepton / 300 μg / was poured into cups, dried for 1 or 2 hours and incubated for 2 or 3 days. Transformants were replicated onto PPMM medium containing thiostrepton / 50 μg / ml / and XP / 30 μg / ml /.

 Transformation of E. coli was carried out according to Cohen et al. / Proc. NAtl. Acad Sci USA. 69: 2110 - 2114, 1972 /.

 Transformants were selected on plates containing ampicillin / 100 μg / ml /. If necessary, X-gal / 36 μg / ml / and IRTG / 10 μg / ml / were added to LA plates.

Hybridization study. DNA transfer from agarose gel to nitrocellulose filters and hybridization was performed as described by Hopwood et al. In the Laboratory Textbook (see above). 7.2 kb Bgl II fragment of plasmid p ULADI and 1 kb. a Bgl II fragment from plasmid p ULAD3 was used as probes. Hybridization was carried out at 70 ^o C for 24 hours. The filters were washed twice for 30 minutes in 2 g of SSC, 0.1% SDS and then two more times again for 30 minutes in 0.2 g of SSC and 0.1% SDS at 70 ^o C.

 Analysis of the nucleotide sequence. The nucleotide sequence was determined by the method of termination of the chain according to Sanger / Sanger and others, Prog. NAtl. Acad Sci. US 74: 5463-5467, 1977. DNA fragments were subcloned into M13mp10 and M13mp11 to obtain insert in any orientation. Ligation mixtures were transfected into competent cells of E. coli strain J M103 and white / hemolytic / plaques were screened for insert insertion. Both strands were sequenced using Amersham International pIc /. United Kingdom / and Sequenases / United States Biochemical Corporation / - analysis kits. All fragments were sequenced using dGTP, but DITP, if necessary, was also used instead of dGTP. The reaction mixtures were separated on 6% or 8% polyacrylamide sequencing gels, which were then transferred onto an x-ray autoradiography film.

 Cloning of promoters. Fragments with activity to initiate transcription were selected using the plasmid of the multicopy promoter probe pIJ 486 - / Ward et al. Mol. Gen. Genet. 203: 468 - 478, 1986 /. Transformants with resistance to kanamycin / Km / were isolated by replicating colonies on MM containing 15 μg / ml Km.

 Transcription-translation in vitro. Plasmids p ULAD 300 and p UC19 were transcribed and translated using a set of laboratory tools for directed translation of prokaryotic DNA / from Amersham Int. PIc./ L / 35S / methionine was used as a radioactive label. For the analysis of labeled proteins, 12.5% polyacrylamide gels containing sodium dodecyl sulfate were used.

 The production of alkaline phosphatase by various streptomyces.

 The production of alkaline phosphatase from ten different streptomyces strains was tested in several solid media. For a list, see table. 1/. S. griseus IMRV3570 and S. griseus ATCC 10137 proved to be the best producers in all tested environments. The best solid medium for the production of alkaline phosphatase was PRMM / containing 30 μg / ml XP /. In this medium, S. griseus IMRU 3570 and S. griseus ATCC 10137 show dark blue staining after 48 hours of growth. S. Lividans 1326 and S. Coelicolor J1 2280 were poor producers of alkaline phosphatase: after 20 hours of growth on PRMM containing XP, was observed only light blue staining.

 Cloning of a gene involved in the production of alkaline phosphatase.

 The total DNA from the ATCC strain 10137 S. griseus was digested with Bgl II, ligated to the digested Bgl II pIJ 702 and the ligation mixture was introduced by transformation into S. Lividans 1326 protoplasts. Transformants were replicated to PRMM containing thiostrepton / 50 μg / ml and XP / 30 μg / ml /. A very dark blue colony was found among 2,800 melanin-negative transformants of S. Lividans. This dark blue colony contained the derivative pIJ 702, carrying 7.2 kb. Bgl II insert: named p ULAD (see FIG. 1).

 Plasmid p ULADI - S. Lividans was unstable and caused white and blue colonies upon transformation. All blue colonies containing the original p ULADI, and white, containing the defective form / with deletion / plasmids p ULADI, were not investigated further. Since instability could be caused by the large insert size in a plasmid such as pIJ 702, which is known for some instability, 7.2 kb insert was subcloned into a 5 kb Bgl II fragment of plasmid pIJ 699 / Kieser et al., Gene 65: 83 - 91, 1988 /. Two plasmids were obtained: p ULAD 100 and p ULAD 101 with insert oriented in opposite directions. Both plasmids were stable and the gene was expressed in both orientations in S. Lividans.

 Localization of the SAF gene.

 7.2 kb The Bgl II fragment was partially digested with a 3A1 and ligated to Bgl II-digested pIJ 702. The ligation mixture was transformed into S. Lividans protoplasts, and the transformants were replicated to PRMM containing XP. Plasmid DNA was isolated from several blue colonies. Four small plasmids were studied in detail: p VLAD2, p VLAD3, p VLAD 16 and p VLAD 18 carrying the determinant of alkaline phosphatase production; these plasmids were stable, being retransformed into S. Lividans prototlasts and carried inserts of 2.4, 1, 2.1, and 1.9 kb / Fig. 1 / respectively. Plasmid p VLAD3 had the smallest 1 kb insert, which could be cut back using Bgl II. When introduced into S. Lividans, pVLAD3 increased alkaline phosphatase production as pVLAD1. pVLAD3 was deposited with depositary number 1-859 05/19/89 in the National Collection of Cultures of Microorganisms, 28th St. Doctors Roux, 175724 Paris, Cedex 15, France, according to the Budapest Treaty and Rule 28 EPC.

 Hybridization of erosomal DNA of several streptomyces.

 Total DNA from strain 10137 S. Griseus; digested BamH1 or Bgl II, hybridized to a 7.2 kb bGl II fragment of pVLAD1 labeled with nick translation with / 32 p / d CTP. Homologous to this probe, a 7.2 kb Bgl II fragment was present in the DNA of ATCC 10137 strain S. Griseus, digested Bgl II, as expected, there was / in this fragment / an internal BamEH1 site that led to two distinct hybridization bands / of 7.9 kb and 9.4 kb when the total DNA was digested with BamH1.

 To determine whether the same gene was present in other streptomyces, total DNA from S. acrimycini, s. coelicolor 1157, s. griseus 2212, s. griseus 3570, s. Lividans 1326 and s. lactamdurans NRRL 3802 was digested with BamH1 and hybridized with a 1 kb Bagl II probe fragment. Hybridization with 9.5 kb with the usual band was observed in the first four above, while 4 and 5 weak hybridization bands were observed in S. Lividans and S. lactamdurans DNA, respectively / Fig. 2 /. In addition, hybridization with DNA of S. albus G, S. coelicolor J1 2280, S. clavuligerus NRRL 3585 and S. fradiae ATCC 10475 was also observed. No further attempts to characterize the hybridization bands were made.

 Incomplete RPO mutants of E. col.

 An attempt was made to establish the complementarity of E. coli phoA mutants E15 and A 1046 when we cloned the alkaline phosphatase structural gene from strain ATCC 10137 S. griseus 7.2 kb Bgl II fragment from pVLAD1 and 1 kb Bgl II fragment from p VLAD3 separately subcloned in both orientations in Bam H1-digested pVC 19. All these plasmid constructs were tested in E. coli M 103 cells and then used to transform E. coli E15 / Sartly et al., J. Bacterial, 145: 288 - 292, 1981 / and E. coli A 1046, no blue colonies were detected in B = XP, suggesting that the complete structural gene for alkaline sphatase is not present in the 7.2 kb fragment of S. griseus, or that this gene is not expressed in E. coli. The structural gene for alkaline phosphatase pho or Bacillus Lichenifotmisi Hullet J. Bacteriol, 158: 978 - 982, 1984 / did not hybridize with the cloned 1 kb fragment / data not shown /. In addition, the phoB / regulatory / E. coli mutant H2 / Kreuzer et al., Genetics 81: 459 - 468, 1975 / was not complemented with either 1 kb Bgl II fragment pVLAD3 or 7.2 Bgl II fragment pVLAD1.

 Superproduction of other extracellular enzymes.

 When pVLAD3 was used for the retransformation of S. Lividans protoplasts, a clear delayed / i.e. delayed / pigmentation and sporulation. These pleiotropic effects have stimulated us to study the role of this gene in protein secretion or in counter-gene expression. As shown in FIG. 3, some extracellular enzymes, including amylase, protease and lipase, were superproduced by S. Lividans transformed with pVLAD3. Beta-galactosidase production also increased, starting about 24 to 30 hours earlier than non-transformed S. coelicolor. Due to the pleiotropic effects of the cloned gene on extracellular enzymes, pigment production and differentiation, this gene was named saf / i.e. activation factor of secondary metabolism.

 Gene dose effect.

 The number of copies of pIJ 702 is estimated to be about 100-200 per chromosome. Although the exact number of copies of pVLAD3 was not determined: the intensity of both bands pIJ 702 and pVLAD3 was not assumed to have a similar number of copies.

 In order to study the effect of gene dosage, we subcloned a 1 kb Bgl II fragment of pVLAD3 into the BamH1 site of plasmid pIJ 61 with a small number of copies / from 3 to 4 copies per cell / / cm. Hopwood et al. Laboratory Textbook. This new plasmid was named pVLAD30. In FIG. 4 shows that the production of extracellular S. Lividans enzymes by transformed pVLAD30 is clearly reduced compared to S. Lividans carrying pVLAD3. In addition, S. Lividans carrying pVLAD30 demonstrated similar pigment production and sporulation patterns as transformed S. Lividans.

 Expression in E. coli and gene trimming.

 Although the pho mutants of E. coli did not complement the saf gene, we observed that 1 kb Bal II insert pVLAD3, being subcloned in one direction in pV C19 /, a plasmid called pVLAD300 / expressed in E. coli, causing abnormal morphology when grown on solid medium, but did not express upon insertion in the opposite direction / plasmid pVLAD302 /. Pazmid pVLAD300 contains ORF / cm. lower / lower from the 1acZ promoter. These results suggest that the saf gene expresses in E. coli from the 1acZ promoter present in p19C19.

 Studies of transcription-translation in E. coli / with plasmid pVLAD300 / were also carried out in vitro, and the reaction products were loaded into a 12.5% polyacrylamide gel. Strip mol. weighing 15,000 was present on the track corresponding to pVLAD300, and was absent in the control track pV C19.

For the exact location of the saf gene, it was decided to continue the cleavage of 1 kb fragment of the plasmid pVLAD300. In these attempts, the existence of unique cut sites for the restriction enzymes SstI / nt216 / Hp h / ht 648 / and Sal GI / nt 936 / within 1 kb fragment was used. Various sub-fragments from a 1 kb insert of pVLAD300 were cloned in the first step into pIJ 2921, the V C18 derivative containing the modified polylinker was flanked by Bgl II sites / Fig. 10 / and then the sticky ends were released using Bgl II and cloned into Bgl II-digested pIJ 702. The production of alkaline phosphatase, amylase and protease was studied with stertomyces Lividans with all plasmid constructs. The results of these studies are shown schematically in FIG. 5, and from the interpretation of these results, the following conclusions can be drawn:
1. 1 kb fragment contains the full saf gene, including its own promoter, since it was expressed at a similar level in both orientations of plasmids pIJ 702 / plasmids pVLAD3 and pVLAD4 /.

 2. This saf gene is probably located in the 648-nucleotide Bgl II-Krp1 fragment / plasmids pVLAD5 and pVLAD6 /.

 3. The Sst1-Kpn1 fragment / 432 nucleotide / probably contains a genetic determinant for superproduction of the extracellular enzyme, but seems to lose its promoter region because it was expressed in only one orientation / plasmid pVLAD14 and pVLAD10 /, but not in the opposite / plasmid pVLAD9 and pVLAD13 /.

 4. The 215-nucleotide fragment of Bgl II-SstI contains the saf gene promoter region.

 5. All effects on extracellular enzymes and on differentiation were preserved and lost together, indicating that the only genetic product is responsible for these actions on all enzymes.

6. Unexpected was the lack of expression of the SstI-Kpn1 fragment (saf gene without promoter), derived from the tyrosinase promoter of the me1 gene present in pIJ 702, which was nevertheless expressed in the opposite direction (clockwise). This discovery implies that there is a fragment with promoter activity located in front of the me1 gene at the Bgl II cloning site / Gi and Hopwood, GENE, 25: 119 - 132, 1983 /. The possibility of functional ORF in the opposite direction / from KpnI-SstI / was dropped for several reasons;
A. Such ORF was not detected by nucleotide sequence analysis.

 B. If such ORF existed, it is clear that it would carry its own promoter, since the Bgl II-Kpn1 fragment was expressed in both directions / plasmids pVLAD5 and pVLAD6 /. In addition, it seems meaningless that the Kpn1 fragment that carries the promoter in the Kpn1 region can be expressed in only one direction.

 C. Expression in E. coli always occurs so that the 1acZ promoter is in front of the putative ORF and never in the opposite direction.

 The promoter activity of the ORF1 fragment of the upper DNA.

 To establish the existence of a promoter in the Bgl II-SstI fragment, from previous experiments it can be concluded that this fragment carries a gene that encodes an aminoglycoside phosphotransferase / neo / without its promoter. Expression of this gene makes S. Lividans resistant to kanamidine and neomycin. When this fragment was subcloned into the pIJ 486 / with the SstI end: closest to the neo gene /, the plasmid pIJ 484: 216 was created. It was observed that S. Lividans transformed with pIJ 486: 216 grows in MM with kanamycin more than 100 μg / ml, whereas S. Lividans transformed with pIJ 486 did not grow on MM supplemented with kanamycin in an amount of 5 mg / ml. These results indicate that such a fragment had promoter activity and supports putative ORF.

Other considerations that are deduced from the nucleotide sequence of probable ORF include:
1. The saf gene is surrounded by two regions with inverted and complementary repeat sequences that can form very stable G = -38.4 Kcal bonds in the mRNA in the presence of the saf gene / nt 637-697 / / fig. eight/.

 2. The sequenced fragment contains a high G + C percentage, which is natural for streptomyces genes.

 3. In front of the saf gene, between ATG / nt 219 / and the repeat structures, there is a very interesting nucleotide region at the top: there are three pairs of repeat nucleotides in it: each with 7 nt / cm. FIG. nine/. It is very likely that this region, especially the promoter region, plays a very important role in the regulation of saf gene expression.

 4. It should be mentioned that the SAF protein contains 18 total positive charges. Based on theoretical principles, this suggests the existence of a significantly greater affinity for DNA. Even in the deduced amino acid sequence, it was possible to observe a very similar region to the regions of DNA-binding proteins (Pobo and Sauer, 1984) (FIG. 7 /.

 The nucleotide sequence of the saf gene.

 The nucleotide sequence of only 1 kb of Bgl II insert pVLAD2 was determined using plasmid pVLAD300 as starting material. Since M13mP10 and M13mp11 do not have a Kpn1 site, fragments with Kpn1 ends were first subcloned into pV C19 and then released as EcoR1-Hind 111 fragments and introduced into mp10 and mp11 digested by EcoR1 and Hind 111. The complete nucleotide sequence of the active region shows ORF1 from 229 nucleotides that encode the putative SAF polypeptide / FIG. 6 /. There is only one possible open reading frame containing a Bgl II-Kpn1 fragment, starting from the ATG initiation codon at nt 183 and ending with the TGA stop codon at nt 524 / ORF1 in FIG. 5 and 6, 6A /. Since the SstI-Kpn1 insert contained in plasmid pVLAD14 showed a lower degree of activity than plasmids also containing the upper region of the SstI site / pVLAD3, pVLAD4, pVLAD5 and pVLAD6 /, it is very likely that ATG / nt 219 / is also in the frame with the hypothesized The saf frame can act as the initiation codon in pVLAD10 and pVLAD14. No other alternative initiation triplets are possible, since a TGA termination codon exists above the first ATG. A long inverted repeat region, which can form a very stable stem / trunk / and loop structure / G = 53.6 Kcal / is present above the saf gene, from nt 90 to nt 135. Another, with a hyphen, inverted repeat sequence was observed down downstream of the termination triplet ORF1, extending from nt 637 to 897 / G = -38.4 Kcal /.

 The saf gene has a high G + C content (78.3%) and labeled genes / Hopwood et al., Regulation of Gene Expression 25 Jearson, Cambridge University Press, pp. 251 - 276, 1986 /.

 The promoter activity of the DNA fragment up from ORF1.

 Since the plasmid lacking the Bgl II-SstI fragment (nt 1 to nt 216) was expressed in only one orientation / plasmids pVLAD9 and pVLAD13 /, it seems likely that the ORF1 promoter was located in this fragment. The presence of a transcription initiation sequence in this region was reinforced by subcloning this fragment into the promoter probe plasmid pIJ 486 / SstI et al., Mol. Gen. Genet 203: 468 - 478, 1986 /, which carried the non-promoter aminoclycoside phosphotransferase / neo gene. Expression of this gene gives S. lividans resistance to kanamycin and neomycin. The Bgl II-SstI fragment was subcloned into the pIJ 486 / SstI end proximal to the neo gene / to obtain a plasmid named pIJ 486: 216. S. Lividans transformed with pIJ 486: 216 could grow on MM containing more than 100 μg / ml kanamycin, whereas S. Lividans, bearing pIJ 486, does not grow on MM with a content of 5 μg / ml kanamycin. This result shows that the Bgl II-SstI fragment has promoter activity. However, the nucleotide homology with other streptomyces promoters did not reveal a typical "consensus" of regions of 10 or 35. / Hopwood et al., Regulation of Gene Expression 25 Jearson, Cambridge University Press, pp. 251 - 276, 1986 /.

 Cloning of the amylase gene.

 The definitive sequence of the amylase gene / amu / S. Griseus 1MPV 3570 has now been explained. The complete nucleotide sequence and the deduced amino acid sequence of the S. griseus amylase gene are shown in FIG. 11. The protein with the leader peptide has 566 amino acids / from NT 318 to NT 2155, both inclusive /, which corresponds to PM 59, 713D. The entire a-amylase gene plasmid pVLTV1 was constructed by digesting S. griseus 1MPV 3570 DNA with 3–9 kb fractions and ligating to Bgl II-digested pIJ 699. After screening for the a-amylase activity of pVLTV1, the plasmid was selected on the content of the whole a-amylase gene. This construction of plasmid pVLTV is given in FIG. 12. Another way to obtain a plasmid with the whole gene of a-amylases is to construct it from two fragments of at least. Among the plasmids obtained by cloning the entire S. griseus DNA library, there are also plasmids that contain a partial gene starting at the 5'-end and those in which the partial gene starts at the 3'-end. From the first named, you can get a 1.3 kb fragment of BamH1-SacI, which includes the 5'-end of the gene, and from the last you can select 1.2 kb SacI-SalI fragment, including the 3'-end of this gene. Both fragments can then be coupled to pIJ 2921 treated with SalI-BamH1 to produce a plasmid with the intact native a-amylase gene / pVLTV200 /.

 Increased saf promoter efficiency.

 In order to test amu gene expression under the control of various promoters, amylase production of 1166 S. Lividans strains was studied. This amugene was placed under the control of various promoters and amylase production was compared with its production using the native promoter / Ramu /. The tested promoters were from the saf / Psaf / gene, from the kanamycin resistance gene / Pneo /, from the PABA / Ppab / synthetase genes, and from the E. coli beta-galactosidase gene / PIae /. Plasmid pVLTV220 containing the amylase gene without promoter was obtained by ligation of the EcoR1-Bgl II fragment isolated from pVLTV200 to plasmid p C18 digested by EcoR1-BamH1, EcoR1-Hind 111, the fragment from pVLTV220 was ligated directly to saf, pab promoters, or (kanamycin resistance) genes to produce pVLTV10, pVLTV A1 and pVLTV80 plasmids, respectively, pVLTV150 containing the 1ac promoter were obtained directly by Hind 111 digesting pVLTV220 and ligating to a 5 kb Hind 111 fragment of pIJ 699.

The study of the production of the obtained plasmids was carried out on plates with MM + starch / 1% /, + thiostrepton, MM / 1 mM P / + starch / 1% / + thiostrepton, MM without phosphate + starch + thiostrepton and MM + starch / 1% / + thiostrepton + 1PTG. Plasmids pIJ 699, pVLTV100 / FIG. 6a / and pVLTV1. A relative measurement of amylase production was carried out by assessing the area of the ring around each colony when staining the growth medium with iodine / 1 ₂ /.

 Production graphs of various designs are shown in FIG. 13. With the exception of control, the highest production was achieved in MM / 1 mM P / + starch + thiostrepton. It is clearly seen that the production peak was obtained by pVLTV10 / which has the Saf gene promoter / - 1482 sq. mm compared to 1335 sq. mm of the other highest producer of pVLTV150, which carries the Plac promoter, when incubated for 114 hours.

 In FIG. 13 / bottom panel, right / pVLVD10 production ratings are presented in comparison with the control under the best known conditions for each type of construction. The increase in pVLVD10 production compared to native pVLTV1 is quite significant: 1482 sq. mm compared to 1105 sq. mm

 Thus, the saf gene promoter can be used as a substitute for other native promoters to improve the expression of various endogenous polypeptides and streptomyces proteins. Similarly, if foreign DNA is inserted, the saf gene promoter is expected to provide similar improved results with amylase, as has been demonstrated.

The expression of a foreign polypeptide or protein
To express a foreign polypeptide or protein, the foreign DNA is preferably inserted into a suitable recognition site for an endogenous gene, preferably a gene encoding an extracellular enzyme, so as to provide secretion of the foreign polypeptide or protein. When this gene is an amylase gene, the BstE11 site is a suitable recognition site / Fig. eleven/. Foreign DNA is insectified, preferably in such a way as to preserve the secretion of signals as much as possible of their extracellular enzyme gene. Since these signals are located mainly on the leader sequence, with respect to the amylase gene, there is evidence that the terminal carboxyl end is also important for secretion. Thus, it may be best to create a cross-linked protein by inserting foreign DNA into endogenous DNA than to remove the transcriptional portion of endogenous DNA and replace it with foreign DNA.

 The sag gene is known to control the production of extracellular enzymes in chromosomal DNA. Chromosomal DNA is believed to have recognition sequences that recognize the SAF polypeptide and cause an increase in extracellular enzyme production. The combination of the saf gene with the amylase gene on the plasmid does not cause increased amylase production.

 Thus, it is preferable to place a foreign protein operably linked to a signal sequence for secretion of an endogenous extracellular enzyme that can be controlled by SAF and, preferably, further operably linked to a saf promoter (in the absence of a native promoter), in the chromosomal DNA as opposed to a plasmid. In this way, the production of a foreign polypeptide a or protein can be further enhanced by native SAF. In another preferred embodiment, the saf gene is inserted through the plasmid into the same organisms in which the foreign DNA has been inserted into the chromosomal DNA. This provides increased secretion of a foreign polypeptide or protein. DNA fragments can be cloned into streptomyces chromosomal DNA using any of the known bacteriophage vectors for streptomyces, such as ⌀ C31 or R4 / Chater et al., Gene Cloning in streptomyces in Gene Cloning in Organisms Other Than E. Coli, published in Berlin, ed. Springer, 1982, pp. 87 - 95 /.

The diagram in FIG. 14 shows a preferred technology for producing foreign protein secretion, in which case the CRF from streptomyces KC400 is a ⌀ C31 derivative that does not have a binding site - / att / and has a C ⁺ gene. See Chater et al., "Gene" 26: 67 - 78/1983 /, the C ⁺ gene encodes a repressor necessary to maintain a lyson state. The att site in the wild-type phage controls the attachment of the phage to the host cell DNA and controls the release from there. Without an att site, this phage cannot be integrated into the host chromosome until a homologous DNA fragment has been cloned into it. Any endogenous gene in the host strain can be used for this purpose and, thus, is simply referred to as the “X gene” in FIG. 14. Phage ⌀ 31 KC 400 processed by known technologies / cm. Hopwood Laboratory Textbook / for insertion of a homologous gene fragment / X gene / as well as a gene including a saf promoter and an endogenous a-amylase gene with a CRF gene operably linked and read to the a-amylase gene. After infection of the host strain of streptomyces, preferably strain S. coelicolor A3 / 2 / available from the John Innes Institute collection. Norwich, UK /, by this phage all phage DNA will be inserted into the chromosomal DNA of the host cell, starting at homologous gene X, preferably using Campbell-type recombination. As indicated, all specific techniques for implementing these procedures are within the knowledge of a person skilled in the art, so that such an insertion can be carried out without undue experimentation.

 These cells with the inserted chromosomal gene are then processed to include the plasmid pVLAD3. Such cells will express the SAF polypeptide, which, in turn, will increase the secretion of a-amylase by the chromosome gene. The presence of the saf promoter on this gene will further increase the secretion of a-amylase. This a-amylase as a secret will contain a CRF molecule attached to it, which can be easily separated by appropriate enzymatic digestion.

 While the streptomyces a-amylase gene has been illustrated by specific examples, it should be borne in mind that in order to increase expression through the use of a saf promoter, the gene used for any polypeptide or protein produced by Streptomyces species can be modified by removing the native promoter and replacing it with saf -promotor. Similarly, a foreign polypeptide or protein can be inserted into any such endogenous gene. It is preferred, however, that the endogenous gene selected by selection is also a gene that expresses the protein through the cell wall and into the culture medium, in which case a secretion signal sequence can be maintained. Best results will be obtained if the gene selected by selection for insertion into a foreign gene is one that is controlled by SAF; and the insertion is carried out in the chromosomal DNA, preferably with simultaneous insertion of the plasmid containing the saf gene.

 While CRF was specifically mentioned, it should be understood that the sequence of foreign DNA can be any DNA sequence that is not derived from streptomyces encoding a protein or polypeptide, in particular of eukaryotic or viral origin. Examples of such eukaryotic and viral DNA sequences are sequences encoding leukocyte interferon of animals and humans / 1FN-a /, fibroblast interferon / 1FN-beta / and immune interferon / 1FN-gamma /, human insulin, human and animal growth and other hormones, such as corticotropin-releasing factor / CRF /, human serum albumin and various human blood factors, and plasminogenic activators - both tissue and urokinase, antigens of the core and surface of the hepatitis B virus, FMD Rusnė antigens and other human, animal and viral polypeptides and proteins.

 While a saf promoter is preferred, the production of a foreign polypeptide or protein will be further enhanced by transforming the host cell with an expression vector containing the saf gene. This expression vector may be located in the plasmid of the DNA of this cell. Thus, an endogenous or any other promoter can be used, and the secretion of a foreign polypeptide and protein will be obtained.

 The previous description of specific implementations so fully reveals the general essence of the invention that others can, using common knowledge in this field, easily modify and / or adapt the invention for its various applications in the form of specific implementations, not departing from the general concept, and therefore such adaptations and modifications are included in the meaning and scope of the disclosed accomplishments. It should also be noted that the phraseology and terminology of this description was not used for the purpose of limitation, but only for general clarity.

 Quantification of carrier protein / amylase / in culture broth / supernatant / cultures of Streptomyces Lividans transformed with various plasmids.

We used three different methods for determining the amount of secreted protein:
1. Spectrodensitometry of protein bands in SDS-PaGE gels stained with silver or photonegatives / films / in the same gels using a Shimadzu spectrodensitometer.

 2. Analysis of the quantities of various proteins using HPLC equipped with a column 08 Aquapor RP-300/200 • 4.6 nm Laboratory Brownley USA /.

 3. Analysis by HPLC equipped with molecular sieves / gel filtration / Protein-pek300 sw columns.

 Results.

 Spores S. Lividans / pVLTV100 /, S. Lividans / pVLVD10, and S. Lividans / pVLTV80 / were inoculated into Leshevalle medium + 1% starch. Maximum amylase activity was obtained, respectively, at 36, 60, 60 h of fermentation of these spores. Total protein in the extracellular fluid / supernatant / was quantified by the Bradford method. 5 μg of protein from each broth was transferred to SDS-PAGE and stained with silver.

 1. Spectrodensitometry was performed on strips in SDS-PAGE / 10% acrylamide / supernatants S. Lividans / pVLTV100 /, 5 μg protein /, S. Lividans / pVLVD10 /, 5 μg protein / and S. Lividans / pVLTV80 / / 5 μg protein /.

 2. HPLC in the column Aquapor RN-300.

 Proteins were eluted with 50 mM acetic acid — acetate with ammonium buffer and a methanol gradient: 0–20 min 0% methanol, 30–35 min 100% methanol, 45 min 0% methanol. Amylase is eluted with 100% methanol for 33 to 34 minutes. The results are shown in table. 3.

 3. HPLC in Protein = Pak300SW.

 Supernatants of culture broths of the same constructs as described above were pre-dialyzed and concentrated by lyophilization. Proteins were eluted with acetic acid ammonium acetate buffer at a flow rate of 0.5 ml / min. Amylase elutes in the form of a peak centered for 21-22 minutes.

Claims (2)

 1. Recombinant PULDVI plasmid DNA for expression of the α-amylase gene in Streptomyces cells, consisting of the vector plasmid pIj 799 and the DNA sequence plasmid built into the Bgl II site, including the SAF promoter with the sequence of FIG. 6 operably linked to the 5'-end of the complete sequence of the amy gene contained in the Hind III 4.8 kb fragment of S. griseus genomic DNA and containing unique restriction sites: Hind III, Nco I, Bst EII, Sal I , Bgl II.  2. The method of expression in cells of the Streptomyces gene. α-amylase, which provides for the isolation of the complete sequence of the α-amylase gene containing the promoter, a region encoding a signal peptide and a sequence encoding the enzyme protein itself, embedding it in a suitable vector, transformation with the recombinant Streptomyces recombinant vector, selection of transformants and culturing them under conditions providing expression of the introduced gene, characterized in that upon insertion into the vector the complete gene sequence or gene sequence devoid of the promoter region is ope affectionately linked to the SAF promoter having the sequence shown in FIG. 6. Priority on points:
05/18/90 according to claim 1;
09/22/89 according to claim 2.

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