FI75367B - FOERFARANDE FOER FRAMSTAELLNING AV EN GENETISKT MANIPULERAD MICROORGANISM INNEHAOLLANDE EN FOER AMYLAS KODANDE REKOMBINANT-DNA, OCH ETT FOERFARANDE FOER FRAMSTAELLNING AV AMYLAS. - Google Patents

Description

1 75367

A method for producing a genetically engineered microorganism comprising recombinant DNA encoding an amylase, and a method for producing an amylase Separated from application 810425.

The invention relates to a method for producing a genetically engineered microorganism containing a gene encoding an amylase, and to a method for producing an amylase using this genetically engineered microorganism.

Although the term genetic engineering is often used to describe a wide variety of methods for artificially altering the genetic information of an organism, it is used in this specification and claims only to refer to the production of recombinant DNA from a donor microorganism and a suitable vector in vitro, selection of desired genetic information. and export of the selected DNA to a suitable microorganism (host microorganism), whereby the desired (foreign) genetic information becomes part of the host's entire genome. The term "cloned microorganism", as used throughout this specification and claims, means a microorganism produced by this method.

The terms "amylase" and "amylolytic enzyme" are synonymous and are used throughout this specification and claims to refer generally to those enzymes capable of catalyzing the hydrolysis of starch, such as alpha-amylase, beta-amylase, isoamylase (alpha-1,6). , glucosidases such as pullulanase, including 30), glucoamylase, etc.

In recent years, of course, much has been written about genetic engineering (two of the many excellent reviews of which are "DNA Cloning and the Analysis of Plasmid Structure and Function," by K.N. Timmis, S.N.

35 Cohen and S.C. Cabello, Prog. Molec. subcell Biol. 6, (1978) 1-58 and "Lambdoid Phages that Simplify the Recovery of 2 75367 In Vitro Recombinants" by Noreen E. Murray, W.J. Bram-mar and K. Murray, Molec. gen. Genet. 150 (1977) 53-6), which contain many descriptions of novel, genetically modified microorganisms with valuable properties. JP Patent No. SHQ 52-76480 by Bunzi Maruo, Yuko Yoneda and Gakuzo Tamura (published June 27, 1997, filed December 19, 1975 as SHO 50-150641) discloses the preparation of amylase-producing microorganism strains in in vivo by ironogenesis, transduction and transformation methods to assemble several genetic traits to promote Amylase production in the Bacillus microorganism. These methods, which do not involve genetic engineering as defined herein, are limited to a single or a few closely related (genetically comprised) microorganisms. These strains are also not suitable for the gene amplification (e.g., phage or plasmid) used in this invention for higher enzyme production. In "Cloning of a Foreign Gene Coding for Alpha-amylase in Bacillus subtilis", Biochemical and Bio-20 Physical Research Communications 91, No. 4, pages 1556-1564 (December 28, 1979), Yuko Yoneda, Scott Graham and Frank E. Young describes the cloning of a gene encoding alpha-amylase into Bacillus subtilis by binding the digested DNA of Bacillus amyloliquefaciens-H to the phi 3T DNA of temperant phage and then trans-forming Bacillus subtilis cells lacking amylase. The authors do not provide any evidence of gene amplification involving uniform production of the amylase enzyme, which is the main object of this invention.

The invention relates to a method for producing a genetically mani-polarized microorganism, in which a recombinant DNA containing a gene encoding an amylase is added to a host microorganism, prepared by an in vitro method by digesting DNA from a donor bacterium and combining the resulting DNA fragments. fragments into a vector similarly digested. The method according to the invention is characterized in that this vector is a plasmid or

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The DNA and genetically engineered microorganism of the 75367 lamda phage derivative is a bacterium capable of producing amylase in a substantially purer form or in a substantially greater amount than that produced by the original donor microorganism under appropriate culture conditions. In a preferred embodiment, the amylases thus produced have one or more properties that make them particularly useful in industrial enzymatic starch hydrolyses (e.g. , dextrose, etc.), such as resistance to high temperatures, heavy metal resistance, etc.

The invention is carried out by first extracting DNA from a bacterial microorganism (donor microorganism) capable of producing at least one amylolytic enzyme, by digesting (with a suitable restriction enzyme) the DNA and, as a vector, the DNA of lambda phage or a derivative thereof and combining and by inserting the resulting fragments to form recombinant DNAs, some of which contain a gene encoding amylase. Recombinant DNAs are rendered biologically active by transfer to appropriate host cells (e.g., E. coli) by in vitro encapsulation or transfection.

The resulting clones are then screened for the gene encoding the amylase, and one or more positive clones are selected and amplified, thereby providing new, genetically engineered microorganisms capable of producing, under appropriate culture conditions, substantially greater amounts of amylase than the donor microorganism. which can be produced. Optionally, the DNA of the new phage is extracted, digested and subcloned into a second vector, which may be either a plasmid or another phage, and the new clones are screened and selected for the presence of the gene encoding the amylase. Sequential "sub-sub-clonings" can also be performed.

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In addition to providing novel, genetically engineered microorganisms that are "overproducers" of amylase in accordance with the invention, the invention has the advantage that it results in the primary transfer of a gene for the production of a single amylolytic enzyme, thus greatly reducing purification, which is necessary in cultures of non-genetically modified micro-organisms.

Once the cloned microorganism containing the desired recombinant DNA has been produced, the microorganism is cultured so that the recombinant DNA is amplified to deliver amylase in substantially greater amounts than can be produced by the donor microorganism.

When the vector is a derivative of lambda phage, in which case the host microorganism is necessarily E. coli, the amplification and production of the enzyme can be carried out as follows.

If the phage is lysogenic, the microorganism, i.e., E. coli, is first cultured to amplify the cells to a suitable density, then infected with an appropriate amount of bacteriophage, and the system is cultured until the cell walls are destroyed and the amylase spreads to the culture medium.

When the vector host system is a suitable lambda-lysogenic E. coli, the infected host microorganism is first cultured at 32 ° C to amplify the bacterial cells to a suitable density, after which the temperature is raised to 42 ° C and maintained there. a period of time to induce a lysogenic cycle and then brought to 37 ° C and maintained there to effect amplification of the foreign DNA present, followed by the production of large amounts of amylase. The cell walls may be destroyed, depending on the conditions, in which case the amylase will spread to the culture medium.

When the vector is a multicopy plasmid, such as pBR 322 or pACYC 184, amplification of the foreign DNA occurs spontaneously. Alternatively, a genetically engineered microorganism containing plasmid DNA is first cultured to amplify bacterial cells to the desired density, followed by the addition of chloramphenicol because the antibiotic inhibits

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7 5 3 6 7 further cell proliferation and amylase production, but allows plasmid DNA to amplify (amplify) within cells. Finally, the cells are separated from the culture medium and washed to remove chloramphenicol. For amylase production, the cells are then re-cultured, of course without chloramphenicol, to produce large amounts of amylase.

Of course, in all genetic manipulations, it is essential to be able to "label" and thus select those clones that contain the desired genetic information.

In the practice of this invention, this is readily accomplished by using a medium containing starch if alpha-amylase, beta-amylase, or glucoamylase activity is desired. The culture medium is then stained with iodine; clones with amylase activity on a starch-containing medium are surrounded by a white region. A specific, preferred staining method is described below. If pullulanase activity is desired, clones are cultured in BBL-trypsinase medium containing pullulan. This method is also described in more detail below.

The invention will now be described in more detail, such as the specific substances and methods used in the work. It should be noted that many of the methods used are standard methods and are well known to those skilled in the art; nevertheless, some of these are described in 25 detail for clarity.

The donor micro-organism

The donor microorganism should be a bacterial microorganism capable of producing at least one Amylase (including, of course, the desired amylase), and preferably produces an amylase having properties desired in the industrial hydrolysis of starch, e.g. resistance to high temperatures or metal poisoning. It is likely that the donor may also be a eukaryotic amylase-producing microorganism (e.g.

55 fungus or yeast). As can be seen from the examples, Bacillus megaterium, Bacillus coagulans, 6 75367

Bacillus cereus and Klebsiella pneumoniae strains to produce cloned microorganisms capable of producing large amounts of alpha-amylase, heat-resistant alpha-amylase, beta-amylase, and pullulanase, respectively.

5 Coupling and Restriction Enzymes T4 DNA ligase has been used consistently as a coupling enzyme in this work, but it is readily understood that any DNA coupling enzyme can be used. The specific restriction enzymes 10 used in this work are shown in the examples. The selection of a suitable restriction enzyme can, of course, be made by one skilled in the art using well known methods. Here, the method for selecting a restriction enzyme for further processing was to digest the DNA extracted from the donor microorganism (donor DNA) with a different restriction enzyme and select the one (or more than one) most suitable for further experiments based on the enzyme's ability to cleave DNA into numerous fragments between 2 -15 kilobases.

Vectors 20 There are several reasons why phage lambda derivatives are particularly effective for cloning DNA extracted from a donor. (1) Phage lambda deletion mutants allow the insertion of foreign DNA fragments of different sizes (depending, of course, on the specific lambda derivative) and, in addition, allow simple identification of clones containing foreign DNA. (2) Various derivatives of phage lambda have been developed that allow the use of several restriction enzymes, thus increasing the chances of successful cloning. (3) Very good amplifications of foreign genes are possible with the use of suitable phage lambda derivatives. (4) After ligation, recombinant clones are easily recovered by transfection or in vitro filling. Due to its versatility, which no other existing vector can provide, this invention is used in the application of phage lambda derivatives and E. coli as a host for the initial cloning of DNA extracted from a donor.

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Other advantages of using phage, rather than plasmid, for primary cloning are as follows: (1) The percentage of recombinants with foreign DNA insertion sequences is higher; (2) The bacteria decompose, thus releasing their cell-5 content and thus all of the amylase into the medium, thus facilitating detection by the iodine staining method; (3) Phage resistance to iodine is higher than to any other living bacterium.

One skilled in the art will readily be able to select a suitable plasmid as a vector for subcloning, one criterion being, of course, the existence of a single or limited number of cleavage sites for the restriction enzyme to be used.

The most widely used plasmids herein are pBR 322 and pACYC 184. In its intact state, plasmid pBR 322 confers resistance to both ampicillin and tetracycline and contains a single cleavage site for each of the Pst I, EcoRI, Hind III, Bam HI and Sal I enzymes. Cleavage and insertion at the Pst I site destroys resistance to ampicillin, whereas insertion into the Bam HI and Sai I sites destroys resistance to tetracycline. Insertion Hind Sweat sometimes destroys resistance to tetracycline, provided that the cloned gene does not have its own promoter.

In its intact state, plasmid pACYC 184 confers 25 resistance to both tetracycline and chloramphenicol, and contains a single restriction site for each of the enzymes Eco R1, Hind III, Bam HI, and Sal I. Cutting and insertion at the Eco RI site destroys resistance to chloramphenicol at three, while Hind III, Bam Hi, and SalI lie have the same effects as plasmid pBR 322 because this region is common to both plasmids.

For subcloning into Bacillus subtilis, the plasmid pC194 vector was used here. This plasmid 35 has a single Hind III site and confers resistance to chloramphenicol.

8 “75367

The host-micro-organism

Since phage lambda derivatives can only be expressed in E. coli, this must obviously be the host for recombinant phage DNA prepared in accordance with this invention. When the recombinant DNA, on the other hand, is in the form of a plasmid, any microorganism capable of accepting and replicating such plasmid DNA can be used, e.g., other bacterial microorganisms or yeasts, such as Saccharomyces cerivisiae.

For practical reasons, E. coli strains (e.g. HB 101) have been used here for the most part because they are well known strains from a genetic point of view and are susceptible to infection with known phages and plasmids, resulting in increased enzyme overproduction capacity.

As described in Example IIa, the recombinant plasmid can be subcloned into a plasmid, such as pC194, which is capable of amplifying in substrate B.

Method

The method is described by a two-step cloning experiment 20, in which phage is used in the first step (batch) and plasmid in the second step.

Once the donor microorganism, restriction and ligation enzymes, and the phage lambda-specific derivative have been selected, the DNAs are extracted, digested, mixed, and the pooled fragments ligated, all by conventional methods not described herein.

The DNA is then made biologically active in E. coli by transfection or in vitro encapsulation. In this work, lambda DNA has been well obtained by the encapsulation method (B. Hohn and K. Murray, Proc. Natl. Acad.

Sci. USA, 74 (1977) 3259-3263). Those clones that have received foreign DNA are then identified by appropriate methods. If an insertion vector such as lambda NM590 or lambda NM607 is used, those clones that contain foreign DNA give clear plaques, while those that do not contain foreign DNA give turbid plaques. When 11,75367 replacement vectors are used, such as lambda NM761 or lambda NM781, identification can be made by culturing an E. coli Lac amber recipient with a lactose indicator medium, e.g., McConkey, EMB, or X gal.

5 Numerous (several thousand) clones that have received foreign DNA are then plated on a medium containing starch and screened for the presence of the gene encoding amylase by the above-mentioned iodine staining method. In this case, of course, care must be taken not to use iodine concentrations so high that the phage are killed, and this can be a problem if iodine is added in the form of a solution. The highly preferred staining method used herein involves exposing the sheets to iodine vapor for a short period of time; this method has been used here with very good success.

A preferred method for finding clones with pullulanase activity that does not use iodine staining is described below. Phages are cultured in petri dishes containing BBL-trypticase and 20 pullulans at a concentration of about 0.25%. Pullulan in the medium is hydrolyzed around the "positive" plaques by pullulanase to maltotriose, which is used by bacteria. Thus, those bacteria that use maltotriosis grow better than others in the medium, with the proviso that plaques that produce pullulanase are surrounded by an opaque ring of growing bacteria and can be easily detected. This method is described in detail in Example IV.

One (or more) positive clones are picked and amplified. This can, of course, be the "final" genetically engineered microorganism and can be used to produce large amounts of amylase by suitable culture, as described above. Alternatively, this clone can be used as a source of DNA for a second cloning into a plasmid or other, more suitable phage. The following describes a method for subcloning into a plasmid.

75367 Again, the DNA of the clones is extracted and digested with a restriction enzyme using standard methods, the plasmid is similarly digested, the fragments are mixed, and the recombinant particles are ligated. Using plasmid pBR 322, the identification of clones containing the gene encoding the amylase is performed by rendering the DNA biologically active in a host, such as by transformation in E. coli, in a culture medium containing ampicillin or tetracycline, depending on the restriction enzyme used. where from. The culture medium also contains starch or pullulan, and clones containing the gene encoding amylase are identified by one of the methods described above. Positive clones are then cultured and plasmid DNA can then be amplified with Chloramphenicol 15 as described above.

The drawings show maps of wild-type bacteriophage lambda (Figure 1a) and all vectors used, as well as new recombinant plasmids produced according to the following examples. In particular, Figure 1 also shows maps of phages from lambda NM 590 (b) and lambda 781 (c), / Murray, N.E., Brammar, W.J.,

Murray, K. Molec. gen. Genet. 150 (1977) 53-6Γ /. Figure 2 comprises maps of plasmids pBR 322 / Bolivar, F., Rodriquez, R.L., Greene, P.J., Betlach, M.C., Heyneker, H.L., 25 Boyer, H.W. Gene 2 (1977) 95-113.7 and pACYC 184 / Chang, A.C.Y., Cohen, S.N. J. Bact. 132 (1978) 1141-1156.7. Figure 3 is a map of the new plasmid pCP 1 and Figure 4 is the new plasmid pCP 2. Figure 5 illustrates Example IIA, showing plasmids pC194, pCP 2.3 and the final new plasmid pCH1.

Figures 6 and 7 show plasmids pCP 3 and pCP 4, respectively. In the maps of all new recombinant plasmids, donor DNA is indicated by a thick line.

The examples illustrate the invention. Percentages are by weight unless otherwise indicated. All experiments 35 were performed following NIH (USA) guidelines for protective structures.

Il 11 75367

Example I

Cloning of the alpha-amylase gene The following substances were used: restriction endonuclease Hind III 5 T4 DNA ligase

Phage lambda NM 590. Phage DNA was prepared by phenol extraction from highly purified phage fragments.

Plasmid pBR 322. Plasmid DNA was purified from lysozyme-digested E. coli cells with cesium chloride-ethidium bromide density gradients.

Host microorganism, E. coli HB 101.

The donor microorganism was a strain of Bacillus megaterium with the following microbiological characteristics: (1) Morphology: rods (0.5 to 0.7 ^ ux 2.0 to 5.0 ^ u), 15 motile, gram-positive, spores are terminal or subterminal.

(2) Restaurant broth: good growth.

(3) Nutritional agar broth: good growth; colonies are dense in the middle and more scattered around.

20 (4) Milk: peptonization without pH change.

(5) Gelatin: not liquefied.

(6) Nitrate reduction: negative.

(7) Catalase reaction: negative.

(8) Oxidase reaction: negative.

25 (9) Cytochrome oxidase reaction: negative.

(10) Indole production: negative.

(11) H2S formation: negative.

(12) Utilization of carbohydrates: use arabinose, xylose, galactose, glucose, levulose, maltose, raffinose, sucrose, starch and acid produced therefrom. Does not utilize rhamnose, mannose, melibiose, inulin and salicin.

(13) Utilization of polyols: utilization of sorbitol and mannitol; does not utilize adenitol, dulcito-35 Ha and inositol.

(14) Decarboxylase reaction with lysine: negative.

(15) Ureolysis: weak.

12 75367 (16) Resistance to heavy metals: increases + + + directly to 500 ppm Cr (17) Sodium chloride broth: increase in NaCl content 3.5%.

5 (18) Optimal temperature for growth: 30-37 ° C

Maximum temperature for growth: 40-50 ° C

Minimum temperature for growth: 5-20 ° C

(19) Oxygen demand: aerobic.

The microorganism was identified as Bacillus megaterium on the basis of its microbiological characteristics with reference to Bergey's Manual of Determinative Bacteriology (8th edition), R.E. Buchanan and N.E. Gibbons, auxiliary suppliers; published by Williams and Wilkins Company, Baltimore, Md. This microorganism is deposited in the National 15 Collection of Industrial Bacteria (NCIB), Torry Research

Station ,. PO Box No. 31, 135 Abbey Road, Aberdeen AB9 8DG, Scotland, 12 February 1980, and has been assigned NCIB No 11568.

The following is a description of the method used.

A. In vitro recombination between lambda and B megaterium-20 DNAs N. 7 yug B. megaterium DNA was digested with 10 units of Hind III at 37 ° C for one hour in 25 yUl of Tris buffer pH 7.5 :in. Approximately 2 μg of lambda NM 590 was similarly digested with the same enzyme. The reactions were stopped by heating at 75 ° C for 10 minutes.

Two tests were performed to determine the efficiency of digestion: 1. A sample of the two reaction mixtures was electrophoresed on an agarose gel for 20 hours at 20 volts. After staining the gel with 30 ethidium bromide, the expected pattern of bands was observed: two bands of lambda DNA derived from the digestion of a single Hind III site with lambda DNA, and a very large number of almost overlapping bands of bacterial DNA digested at numerous sites. .

35 2. The transfection assay of phage DNA showed that digestion was more than 99% efficient, thus almost completely eliminating the biological activity of the phage DNA.

13 75367

The digested DNAs were mixed and incubated for five hours at 10 ° C with 0.15 units of T4 DNA ligase to allow arbitrary reheating and covalent binding of the DNA fragments.

At the end of this incubation, the DNA was mixed with an in vitro encapsulated preparation comprising a mixture of two complementarily deficient phage lysates (lambda Dam and lambda Earn) induced from non-suppressive strains. In this mixture, any DNA molecule having the cos-extreme tails of lambda 10 DNA and of a suitable size can combine with a phage protein, thus forming an in vitro biologically active phage particle capable of infecting E. coli and producing an infection center (plaque).

To determine the efficiency of this process, two checks were performed: 1. A sample of the mixture was applied to E. coli, 3 x 10 plaques per μg of lambda DNA used were found. This was about 100 times more than was found in the digested preparation, indicating good efficiency of the ligase treatment. 20 2. The plaques thus formed were examined visually. 70% of them were clear instead of turbid, indicating the presence of a fragment of B. megaterium DNA associated with lambdagen Cl, and thus inactivating it, causing plaque turbidity.

Thus, it was found that the preparation contained a blank sample of all Hind III DNA fragments possible for B. megaterium that could be inserted into phage lambda NM 590.

Isolation of a phage lambda derivative with B. megaterium 30 amylase

The remainder of the encapsulation mixture was used to inoculate E. coli with a large number of starch-containing plates 3 with about 10 viable particles per plate. After growth of infected E. coli and plaque formation, the plates were screened for the presence of starch-degrading plaques. This was done by exposing the plates to iodine 75367 vapors for a short time; it was expected that the plaque formed by such a phage would be surrounded by a clear region as a result of the diffusion and function of the amylase released from the lysed cells. One such plaque was found and the phage 5 it contained were immediately picked up for subculture (prolonged exposure to iodine vapors would have killed the phage). This plaque multiplied really abundantly (producing amylase) and is referred to herein as "lambda NM 509 Amy l". Phage lambda NM 590 Amy 1 is stored in the collection on 12.12.1980 as NCIB 10 No. 11569.

C. Recloning of the amylase gene into plasmid pBR 322 This was performed to obtain an overproducing strain, it was also intended to prepare a large amount of DNA containing the amylase gene.

15 μg of DNA from lambda NM 590 Amy 1 and 0.3 μg of plasmid pBR 322 DNA were digested, mixed and treated with ligase as described in Part A. This preparation was used to transform (using standard methods) strain HB 101. Selection was for ampicillin (ap) -20 resistance (a property that cells receive from the presence of this plasmid). This plasmid normally also confers resistance to tetracycline (Te). However, the introduction of foreign DNA into this plasmid cleaves the tet gene, thus the proportion of tetracycline-sensitive transformed clones is reflected in the proportion of plasmids containing cloned DNA. In this case, 16% of the clones contained a new plasmid that gave amylase activity to E. coli. According to the General International Nomenclature of Plasmids, the plasmids described herein are designated (pCP) and the specific plasmid of this example is designated (pCP1). This indicates that recloning of the gene by the same enzyme (in this case Hind III) is very efficient method (16% instead of 10 ^).

The microorganism containing this plasmid is designated 35 E. coli Cl 7001 (pCP 1) and was deposited with the NCIB on February 12, 1980 as NCIB No. 11570.

Il 15 75367 D. Amplification and enzyme production

Enzyme production in plasmid (pCP 1) -containing cells was enhanced in two ways as follows: 1. Saturated cultures 5 The culture was incubated overnight at 37 ° C with a rotary shaker in 5 L septum Erlenmeyer flasks containing 1 liter of culture medium (LB; ).

2. Amplification with chloramphenicol 10 The culture was incubated at 37 ° C on a rotary shaker in 5 l septum Erlenmeyer flasks containing 1 liter of culture medium (LB) until a density of 0.8 (650 nm) was reached, at which point 150 ug / ml chloramphenicol. This prevented further amplification of chromo-15 somatic DNA, but not amplification of the plasmid containing the amylase gene (pCP 1). After amplification of the plasmid (up to 3,000 copies) compared to chromosomal DNA, chloramphenicol was removed to allow resumption of protein synthesis. Specifically, after amplification, cells were separated from the medium by centrifugation, washed to remove chloramphenicol, and re-cultured for amylase production.

E. Enzyme Recovery The "osmotic shock" method was used to recover 25 alpha-amylase in high purity. The method presented by H.C. Neu and L.A. Heppel in J. Biol. Chem. 240 (1965) 3685-3692 was as follows:

The cells were first cultured overnight, then suspended in 25% sucrose solution, half the volume of the culture, and shaken for 10 minutes at 24 ° C, which plasmolyzed the cells. EDTA was then finally added to 1 mM (to permeate the cell walls), and the material was shaken for another 10 minutes to 35 ° C, the suspension was centrifuged, and the cells were rapidly resuspended in cold (ca. 0 ° C) water. and shaken at this temperature for 10 minutes. The suspension was resuspended and 96% of the enzyme could be recovered from the supernatant.

For comparison, the donor microorganism (Bacillus megaterium) was cultured and the enzymatic activity was determined. The amount of enzyme per milliliter of culture medium was measured by the DNA method, where one enzymatic unit was defined as the amount of enzyme that produces one mg of reducing sugar (using maltose as a reference) per minute over a 10 minute incubation period. The substrate was very pure amylose.

The donor microorganism produced 66.0 enzymatic units / l of culture. Saturated cultures of E. coli CL 7001 (pCP 1) produced 116.6 units / l of culture, whereas after culturing (confirmation) for 5 hours with chloramphenicol, followed by 15 hours without chloramphenicol, E. coli CL 7001 (pCP 1) produced 84.5 units / liter. This indicates that the impregnated culture method is sufficient to provide a good increase in enzyme production.

The enzyme produced by E. coli CL 7001 (pCP 1), which has been identified as an alpha-amylase, has the following characteristics. It cleaves both amylose and amyl-25 pectin into glucose, maltose and maltotriose, mainly maltose, and cleaves both cyclodextrin and maltotriose into glucose and maltose. It breaks down pullulan into a panose and / or iso-panose, a property similar to that of the enzyme recently described by Mizucho Shimizy, Mutsuo Kanno, Masaki Tamura and Mikio Suekane "Purification and Some properties of a Novel Alpha-Amylase Produced by a Strain of Thermoactino-myces vulgaris ", Agric. Biol. Chem. 42 (1978) 9, 1681-1688.

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17 75367

Example II

Cloning of the heat-resistant alpha-amylase gene

The donor microorganism was the original Bacillus strain isolated from the compost and identified as 5 Bacillus coagulans. It produces heat-resistant alpha-amylase and has the following microbiological characteristics: (1) Morphology: rods (0.6 to 1 μl x 2.5 to 5.0 μm), mobile gram-positive and -negative. The spores are 10 central or terminal, do not change shape.

(2) Restaurant broth: good growth.

(3) Nutritional agar incision: good growth, filamentous, spreading, creamy white.

(4) Utilization of organic acid 15 - citrate: positive - malonate: negative.

(5) Gelatin: liquefied (6) Production of acetylmethylcarbinol (acetane): positive.

(7) Orthonitrophenyl-galactoside hydrolysis: positive.

(8) Nitrate reduction: positive, gas may be formed.

(9) Catalase reaction: positive.

25 (10) Indole production: negative.

(11) Decarboxylase reaction - with lysine: negative - with ornithine: negative - with arginine: negative.

30 (12) H2S formation: negative.

(13) Utilization of carbohydrates: utilizes arabinose, galactose, glucose, levulose, mannose, maltose, sucrose, starch, trehalose and produces acid from them.

35 (14) Pullulan is utilized with a minimum medium.

18 75367 (15) Utilization of polyols: utilizes glycerol, sorbitol, mannitol, inositol. Does not use Adoni tolol, dulsitol.

(16) Ureolysis: negative.

5 (17) Utilization of lecithin: negative.

(18) Optimum temperature for growth: 50 ° C

Maximum growth temperature: 55-60 ° C Minimum growth temperature: 15-25 ° C

(19) Oxygen demand: aerobic and anaerobic.

10 The stock was deposited in the NCIB collection on 21 February 1980 NCIB

No. 11571.

DNA was extracted and exposed to Eco RI; Price III: n;

Pst I: n; Sal I: n; Bam HI and Bgl II. Only Bgl II was able to generate several fragments from a wide range of 15 molecular weights. However, it was possible to generate fragments with Eco R1 when the NaCl concentration was reduced to 50 mM. Therefore, it was decided to use Eco R1 to generate fragments for cloning into the Eco RI lambda vector (lambda NM 781).

Digestion of 20 AB coagulans and lambda NM 781 DNAs 1.25 ug of lambda NM 781 DNA was cut with one unit of Eco R1 in 25 of the following buffer: 10 mM Tris HCl (pH 7 , 5), 10 mM 2-mercaptoethanol, 10 mM MgSO 4 and 100 mM NaCl.

2 .mu.g of B. coagulans DNA was cut with the same enzyme in a similar buffer, except that the NaCl concentration was reduced to 50 mM. The incubation time was two hours at 37 ° C and the reaction was stopped by heating for 10 minutes to 75 ° C. The completeness of the digestion was checked by electrophoresis on 1% agarose gels.

Bi Recombinant phage ligation and recovery

The digested DNAs were mixed and ligated using two units of T4 DNA ligase in a mixture of 60 mM Tris HCl (pH 8), 10 mM MgSO 4, 10 mM 2-mercaptoethanol and 0.1 mM ATP. The reaction was allowed to proceed for 10-15 hours at 10 ° C. After ligation, 0.2 μg was mixed

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19 75367 -2 DNA batches with ATP to give a final concentration of 10 M. These samples were subjected to in vitro encapsulation and used 4 to infect E. coli strain HB 101. Approximately 1.6 x 10 PFU (Plaque Forming Units) was obtained. units-5) / yug of lambda DNA. Some of these phages showed amylase activity (for detection of amylase activity in petri dishes and for phage recovery and purification, see Example I).

A fairly high proportion of amylase-producing phages 10 (one in 400) was observed.

One was selected and named "lambda NM 781 alpha Amy 1". It was deposited in the NCIB collection on 12 February 1980 as NCIB No. 11572.

C. Recloning of the Amylase Gene from lambda NM 781 alpha Amy 1 to Plasmid pBR 322 1 μg of lambda NM 781 alpha Amy 1 DNA and the same amount of DNA from plasmid pBR 322 were cut with Eco R1 using standard conditions. Ligation and transformation were performed as described in Example I. Colonies of E. coli HB 101 containing the recombinant plasmid were detected for their amylase activity. One such colony was selected and named E. coli CL 7002 (pCP 2). It was deposited in the NCIB collection on 12 February 1980 as No 11573.

D. Amplification of the Gene Product 25 Confirmation of the gene encoding amylase and amylase production of Lambda NM 781 alpha Amy 1 was performed as follows. Host bacteria (E. coli HB 101) were grown in LB medium with 2 mM MgCl 2 at 37 ° C until an optical density of 0.3 (650 nm) was reached. Lambda NM 781 30 alpha Amy 1 was then added, the number of phages was counted to obtain a multiplicity of infection of about 1-2, i.e. one or two phages per bacterial cell. The culture was then continued with vigorous stirring at 37 ° C and the optical density was monitored until it began to decrease. When it had fallen below 0.5, the culture was harvested and placed on ice. The culture was centrifuged and the supernatant was tested for amylase activity.

--------. .. 1.

20 75367

Amplification and enzyme production of E. coli Cl 7002 (pCP 2) was performed using both a saturated culture method and chloramphenicol amplification as in Example I.

Amylase activity was determined using a DNA method from both phage lysate and cultures of E. coli containing the recombinant plasmid. Table I shows the values obtained for amylase activity in the original B. coagulans strain and the distribution between extracellular and so-10 bone-bound activity. Activities obtained with recombinant phage (lambda NM 781 alpha Amy 1) and a strain containing the recombinant plasmid E. coli CL 7002 (pCP 2) are also given. Approximately a three-fold increase in enzyme production was observed with the recombinant phage, and a much better increase was observed with the plasmid (about 300-fold).

In the case of phage (lambda NM 781 alpha Amy 1), all the enzyme is released in cell lysis and is thus present in the culture medium. In the case of the plasmid, most of the enzyme is bound to the cell (96%).

E. Recloning into a phage lambda derivative capable of lysogenizing E. coli

To illustrate the preparation of a vector / host system comprising lambda lysogenic E. coli, the following experiment was performed.

Bacteriophage was used for lambda T4 liq. CI 857 Warn Ean Sam (lambda NM 989). It is described in J. Mol. Biol.

Butter. 132 (1979), "Molecular Cloning of the DNA Ligase Gene from Bacteriophage T4. 1. Characterization of the Recombinants 30" G.G. Wilson and Noreen E. Murray (pp. 471-491) and "II .. Amplification and Separation of the Gene Product" Noreen E. Murray, S.A. Bruce and K. Murray (pp. 493-505).

This phage contains the bacteriophage T4 DNA ligase-35 gene and is able to lysogenize E. coli. It also has a heat-sensitive immunity gene and two amber mutations in the E gene and the S gene, respectively. A mutation in the S gene 21 75367 makes the bacteria no longer degrade by infection with this phage. The purpose of subcloning was to replace the DNA ligase gene with a gene encoding amylase.

Phage and plasmid (pCP 2) DNA were digested with Eco R1 and fragments were ligated. The phage DNA obtained in the ligation was then packaged in vitro and the plaques were visualized by plating E. coli Sup E Sup F. Plaques with amylase activity were picked and the phages were purified using the methods described above.

This phage was then used to lysogenize strain E. coli C 600 (CL 1205) using conventional methods. Lysogen colonies were visualized with starch-containing medium using iodine staining. This strain is designated herein as E. coli CL 7003 (lambda alpha Amy 1) and was deposited with the NCIB on March 6, 1980 as NCIB No. 11586.

Airplication of the gene product was performed as follows. Lysogen was cultured at 32 ° C in LB medium until a density of 0.8 at 650 nm was reached, then centrifuged and the cells were suspended in fresh LB medium preheated at 45 ° C. The culture was then incubated in a 45 ° C bath for 15 minutes to induce a digestion cycle (because the immune gene product is heat sensitive).

The incubation was then continued with vigorous shaking at 37 ° C for three hours. Amylase activity was then measured in the supernatant and cells. The values are shown in Table I.

This special experiment illustrates the "sub-sub-cloning" method from plasmid to new lambda phage. It is readily understood that DNA from lambda NM 781 alpha Amy 1 could just as easily have been sub-cloned into phage lambda T4 lig. CI 857 Warn Earn Sam. Alternatively, the donor DNA could have been cloned directly into the latter phage, with any number of modifications from the exemplary process being feasible.

22 7 5 3 6 7 F. Recovery and characterization of amylase produced by E. coli CL 7002 (pCP 2) and E. coli CL 7003 (lambda alpha Amy 1) by lambda NM 781 alpha Amy 1

As in Example I, osmotic shock was used to recover the 5 enzymes. In addition, since the enzyme of this example is heat resistant, it could be further purified by adding 10 mM Ca ++ to the aqueous solution and heating the solution to 80 ° C and keeping it there for 10 minutes; this treatment precipitates the recovery of all E. coli proteins and sal-10 lii alpha-amylase in an extremely pure form, in which case no organic waste or other enzymes are usually present.

The specificity of alpha-amylase was determined: it is active against amylose and starch, the hydrolysis products are glucose, maltose and maltotriose, and small amounts of higher molecular weight components. This enzyme is not active for cyclodextrin. The heat resistance of the enzyme was also studied and shown to be very high. The optimum temperature for activity with 0.5% amylose is between 80-90 ° C. For 8% of soluble starch, the optimum is around about 100 ° C.

It is possible that the microorganism referred to herein as Bacillus coagulans is in fact Bacillus licheniformis.

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75367 24

Example IIA

Subcloning of plasmid pCP 2 into plasmid pc 194 and expression of the new plasmid in Bacillus subtilis.

This example illustrates a method by which recombinant DNA prepared according to the invention can be expressed in a host bacterium other than E. coli, namely a strain of Bacillus subtilis *

Plasmid pCP 2, from Example II, contains 3.31 Kb fragments from a B. coagulans donor; the plasmid is further characterized by conferring ampicillin and tetracycline resistance and containing two cleavage sites for both Eco R1 and Hind III. Plasmid pCP 2 was digested into four fragments of both Eco R1 and Hind III, treated with ligase, and used to transform HB 101, 15 as in the previous examples.

New plasmids, designated herein as pCP 2.3, were constructed with 2.64 Kb fragments of B. coagulans DNA, this fragment containing the gene encoding alpha-amylase. pCP 2,3 is a single site for both Eco R12 and Hind III and confers both ampicillin and tetracycline resistance.

For the next step, plasmid pC 194, which is known to be able to amplify in Bacillus subtilis, was selected. pC 194 gives the intensity of chloramphenicol resistance-25 and has a single Hind III site.

Both pCP 2,3 and pC 194 were opened with Hind III, mixed and ligated to form a new plasmid, designated pCH 1, which contained the gene encoding alpha-amylase and had both chloramphenicol resistance 30 and ampillin resistance, albeit chloramphenicol resistance. tensile levels were low in E. coli. As above, the preparation was used to transform E. coli HB 101.

B. a mutant strain of subtilis designated QB 1133 (obtained from Cr Michael Steinmetz, Institut de 35 Recherche en Biologia Moleculaire, Universite Paris VII,

Tour 43, 2 Place Jussieu, 75221 Paris Cedex 05), and lacking alpha-amylase activity, was then treated with 75 757 S. Chamg and S. Cohen, Molec. Gen. Genet. 168 (1979)

H1-115 according to the method for forming protoplasts. The protoplasts were then transformed with pCH 1 using the polyethylene glycol-mediated transformation method of Chang and Cohen (Ibid).

The protoplasts were then incubated in rich medium for 1.5 hours to allow the plasmid in the bacteria to express resistance to chloramphenicol.

The protoplasts were then applied to regeneration medium supplemented with chloramphenicol at 20 ug / ml. After two days of incubation at 37 ° C, colonies of transformed cells had appeared; colonies with alpha-amylase activity were detected by the iodine vapor staining method. One clone, designated B. subtilis 15 CL 8001 (pCH 1), was deposited on January 13, 1931 as NCIB No. 11629. The original strain QB 1133 was also deposited on 13 January 1981 as NCIB No. 11628.

B, subtilis CL 8001 (pCH 1) is stable only in the presence of chloramphenicol. It can be used to produce alpha-amylase by culturing in a medium containing chloramphenicol (20 μg / ml). Alpha-amylase can be easily recovered from the culture medium.

After overnight culture in the presence of chloramphenicol, the amount of alpha-amylase produced was determined as follows: Upper liquid Cells Total (U / L) (U / L) (U / L) 136 14 150

Example III

30 Cloning of beta-amylase

The donor microorganism was a strain of Bacillus cereus described in British Patent No. 1,466,009 to the Agency of Industrial Science & Technology (Japan). It was deposited in Fermentation 35 Research Institute of Chiba-shi, Japan on December 20, 1973 FERM - P No. 2391 and also deposited in the American Type Culture Collection ATCC No. 31102 on December 26, 1974. Its 26 7 5 3 6 7 is known to produce both beta-amylase and alpha-1,6-Glu cosidase.

A. Cloning of the beta-amylase gene in lambda NM 781

The methods used for digestion, ligation, and recovery of recombinant phages were the same as in Example II. B. cereus DNA (2 μg) was digested under normal conditions with the restriction enzyme Eco R1. Phage vector DNA (1.25 μg, lambda NM 781) was also cut with Eco R1. Ligation and packaging were performed as described above.

Several recombinant phages with amylase activity (about 1,500) were found. One of these phages (designated "lambda NM 781 beta Amy 1") was selected and deposited in the NCIB on February 12, 1980 as NCIB No. 11574.

B. Recloning of the lambda NM 781 beta-amylase gene from beta Amy 1 into plasmid pBR 322

To increase enzyme production, this first beta-amylase clone was used as a source of DNA encoding beta-amylase to subclon it into multicopy plasmid pBR 322.

2 μg of lambda NM 781 beta Amy 1 DNA and 1 μg of pBR 322 were cut with Eco R 1, mixed and treated with T4 ligase. Ligation and transformation were performed as described above.

One of the 200 ampicillin-resistant colonies had starch degrading activity.

One was isolated and designated E. coli CL 7004 (pCP 3); it was deposited on 15 September 1980 as NCIB No. 11602.

C. Recloning of the Beta-Amylase Gene into a Phage 30 Lambda Derivative Capable of Lysogenizing E. coli The vector used was heat-sensitive lambda T4 lig phage CI 857 Warn Earn Sam (lambda NM 989) described in Example II. Approximately 1 ug of plasmid pCP 3 DNA and 0.5 ug of phage DNA were digested, then the fragments were ligated together and the resulting DNA fragments were packaged in vitro. Phage particles with amylase activity were isolated and used to give lysogenic colonies by the same method as above.

Il 27 75 3 67 E. coli strain C 600, which is lysogenic for this recombinant phage, was isolated and designated E. coli CL 7005 (lambda beta Amy 1); this was deposited on 15 September 1980 as NCIB No. 11603.

5 D. Amylase gene product amplification

Amylase activity was detected in all clones and subclones by DNA method. Beta-amylase activity was confirmed by TLC chromatograms in the presence of a single spot of maltose after amylose digestion.

Amplification of the beta-amylase gene in different clones was performed as described above. Table II shows the enzymatic activity of the original strain-15 sa compared to this activity in three clones; this activity was obtained either from the phage lysate or by the osmotic shock method described in Example II.

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Example IV

Cloning of the pullulanase gene

The donor microorganism was Klebsiella pneumoniae, described by H. Bender in Biochem. Z. 334 (1961) 5 79-95 and deposited with the ATCC on June 6, 1983 as No. 15050. It is also mentioned in A.E. Staley's British Patent No. 1,273,789.

2.25 ug of donor DNA was extracted and fragmented with Eco RI using standard conditions, and 1.25 ug of lambda 10 NM 781 DNA was digested with the same enzyme under the same conditions. The incubation lasted for three hours at 37 ° C, after which the reaction was stopped by heating for 10 minutes at 75 ° C. The completeness of the cleavage was determined as in Example lii

The digested DNAs were ligated, recovered, and used to infect E. coli strain HB 101, all as in Example II. Approximately 2 x 10 10 PFU / μg lambda DNA was obtained.

After two days of incubation at 37 ° C in plates containing BBL-trypticase + 0.25% pullulan, some plaques showed pullulanase activity, i.

20 they are surrounded by turbid rings characteristic of “overgrown” bacteria.The proportion of phagulanase-producing phages was on the order of 1/2500.

One was selected and named here lambda NM 781 Pui 1; it was deposited in the NCIB on April 9, 1980 as NCIB 25 No. 11592.

Pullulanase activity was detected by DNS and the product of hydrolysis of pullulan by phage lysates was identified as maltotriose by thin layer chromatography.

Lambda NM 781 Pul 1 contains a large DNA fragment 30 (Klebsiella pneumoniaesta) 13.5 Kb in length; this fragment was re-digested with Eco R1, resulting in two fragments, 6.1 Kb in length and 7.4 Kb, respectively.

The 6.1 Kb subfragment was then recloned into lambda NM 781 and shown to contain the pullulanase gene. 35 This new recombinant phage is named lambda 781 Pui 2; it was deposited on 15 September 1980 as NCIB No. 11604.

The 30 7 5 3 6 7 6,1 Kb subgroup was further subcloned into a multivariate

in the pioplasmid pACYC 184 (a derivative of pBR 322 with R R

the Te gene of the latter and the Cm gene with one Eco RI site). This new clone is designated E. coli CL 7006 5 (pCP 4); it was deposited on 15 September 1980 as NCIB No. 11605.

A third subclone was constructed by ligating a 6.1 Kb fragment into phage lambda NM 898 as described in Example II. This clone is designated E. coli CL 7007 (lambda Pui 2); it was deposited on 15 September 1980 as NCIB No. 11606.

The level of pullulanase expression and amplification was examined in all clones. The results are summarized in Table III.

As can be seen from the results, maltose induces pullulanase activity when 0.04% maltose is added to 15 LB medium. In addition, Triton X 100 treatment of the membrane fraction was necessary to recover pullulanase, indicating that the activity is located in the membranes.

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Claims (18)

32 Claims 7 5 3 6 7 A method for producing a genetically engineered microorganism, comprising adding to the host microorganism a recombinant DNA comprising a gene encoding an amylase prepared by an in vitro method by digesting DNA from a donor bacterium and combining the resulting DNA fragments into a vector which is simultaneously cleaved, characterized in that the vector is a plasmid or a DNA of a lamdba phage derivative and the genetically engineered microorganism is a bacterium which, under suitable culture conditions, is capable of producing amylase in a substantially purer form or in a substantially greater amount than the original donor microorganism. Method according to Claim 1, characterized in that the donor bacterium is a strain of the genus Bacillus. Method according to Claim 1, characterized in that the donor bacterium is Bacillus megaterium, Bacillus coagulans, Bacillus cereus or Klebsiella pneumoniae. Method according to Claim 1 or 3, characterized in that the donor bacterium is Bacillus megaterium NCIB No 11568, Bacillus coagulans NCIB No 25 11571, Bacillus cereus ATCC No 31102 or Klebsiella pneu monoge ATCC No 15050. Method according to one of Claims 1 to 4, characterized in that the vector is plasmid pBR 322, pACYC 184 or pC 194. Method according to one of Claims 1 to 4, characterized in that the vector is phage lambda NM 590, lambda NM 781 or lambda NM 989. Method according to one of Claims 1 to 4 or 6, characterized in that the recombinant DNA comprises 35 phages lambda NM 590 Amy 1, NCIB No. 11569; lambda NM 781 alpha Amy 1, NCIB No. 11572; lambda NM 781 beta Amy 1, NCIB 33 7 5 3 6 7 No. 11574; lambda NM 781 Pul 1, NCIB No. 11593 and lambda NM 781 Pul 2, NCIB No. 11604. Method according to one of Claims 1 to 5, characterized in that the genetically engineered microorganism is E. coli or B. subtilis. Method according to one of Claims 1 to 5, characterized in that the genetically engineered microorganism is E. coli CL 7001 (pCP 1), NCIB No. 11570; E. coli CL 7002 (pCP 2), NCIB No. 11573; E. coli CL 7003 (lambda alpha A method for producing an amylase, characterized in that the genetically engineered microorganism prepared according to any one of claims 1 to 9 is cultured under amylase-producing conditions. 10 Amy 1), NCIB No. 11586; E. coli CL 7004 (pCP 3), NCIB No. 11602; E. coli CL 7005 (lambda beta Amy 1), NCIB No. 11603; E. coli CL 7006 (pCP 4), NCIB No. 11605, E. coli CL 7007 (lambda Pui 2), NCIB No. 11606 or B. subtilis CL 8001 (pCH 1), NCIB No. 11629. The method according to claim 10, characterized in that the recombinant DNA is present in the form of disruptive phage and the culture is performed in an infected bacterial host. Method according to Claim 10, characterized in that the recombinant DNA is present in the form of phage in lysogenic E. coli and the culture is carried out in such a way that phage amplification is induced by heat treatment. A method according to claim 10, characterized in that the recombinant DNA is present in the genetically engineered microorganism in the form of a plasmid and the culture is carried out up to the stationary phase. A method according to claim 13, characterized in that the plasmid is a derivative of pBR 322 or pACYC 184 and amplification is enhanced by culturing the microorganism in the presence of chloramphenicol before culturing it to the stationary phase. 34 75367 The method according to claim 10, characterized in that the amylase thus prepared is alpha-Amylase and the recombinant DNA of the microorganism contains a gene encoding amylase obtained from Bacillus megaterium. The method according to claim 10, characterized in that the amylase thus prepared is a heat-resistant alpha-amylase and the recombinant DNA of the microorganism contains a gene encoding amylase obtained from Bacillus coagulans. The method according to claim 10, characterized in that the amylase thus prepared is a beta-amylase and the recombinant DNA of the microorganism contains a gene encoding amylase obtained from Bacillus cereus. The method according to claim 10, characterized in that the amylase thus prepared is a pullulanase and the recombinant DNA of the microorganism contains a gene encoding amylase obtained from Klebsiella pneumoniae. Il 35 Patentkrav 7 5 3 6 7