EP0672154A1

EP0672154A1 - A PROCESS FOR EXPRESSING GENES IN $i(BACILLUS LICHENIFORMIS)

Info

Publication number: EP0672154A1
Application number: EP92923721A
Authority: EP
Inventors: Steen Troels Jorgensen; Per Lina Jorgensen
Original assignee: Novo Nordisk AS
Current assignee: Novo Nordisk AS
Priority date: 1991-11-14
Filing date: 1992-11-13
Publication date: 1995-09-20
Also published as: JPH07503363A; FI942227A; WO1993010248A1; FI942227A0

Abstract

A process for expressing genes derived from anaerobic and/or thermophilic microorganisms in Bacillus licheniformis, in which process a suitable strain of B. licheniformis transformed with a DNA sequence which includes a gene derived from an anaerobic and/or thermophilic microorganism, which DNA sequence is preceded by a promoter sequence capable of effecting transcription of said gene, is cultured under suitable conditions to obtain gene expression.

Description

A PROCESS FOR EXPRESSING GENES IN BACILLUS LICHENIFORMIS

FIELD OF INVENTION .

The present invention relates to a process for expressing genes derived from anaerobic and/or thermophilic microorganisms in Bacillus licheniformis, as well as to a process for producing cyclodextrin glycosyl transferase in Bacillus licheniformis.

BACKGROUND OF THE INVENTION

Cyclodextrin glycosyl transferases (1,4-α-D-glucan 4-α:-D-(l,4- α-D-glucano)transferase, EC 2.4.1.19), hereinafter termed CGTases, have previously been employed in the liquefaction of starch or starch hydrolysate, and for the formation of cyclodextrins by cyclisation. The CGTases so far used for this purpose are produced by such microorganisms as Bacillus macerans, Bacillus circulans. Bacillus stearothermophilus, Bacillus megaterium. Bacillus ohbensis, alkalophilic Bacillus sp. , Micrococcus luteus, Micrococcus varians and Klebsiella pneumoniae. These CGTases suffer from the disadvantage that they are not sufficiently stable at temperatures above 60°C to be useful in the production of cyclodextrins at sufficiently elevated temperatures to avoid microbial contamination. More recently, CGTases derived from a strain of Thermoanaerobacter or Thermoanaerobium have been isolated, as described in WO 89/03421. These CGTases have a temperature optimum at pH 5.0 of about 95°C.

For the production of large amounts of CGTase, it is an advantage to produce it by recombinant DNA techniques. The cloning of genes encoding a CGTase derived from EL. macerans or B. stearothermophilus has been described in GB 2 169 902. The cloning of the gene coding for the Thermoanaerobacter sp. CGTase in _£_,_ coli is described in WO 89/03421. SUMMARY OF THE INVENTION

The present invention relates to a process for expressing genes derived from anaerobic and/or thermophilic microorganisms in Bacillus licheniformis. in which process a suitable strain of B_i_ licheniformis transformed with a DNA sequence which includes a gene derived from an anaerobic and/or thermophilic microorganism, which DNA sequence is preceded by a promoter sequence capable of effecting transcription of said gene, is cultured under suitable conditions to obtain gene expression.

In another aspect, the present invention relates to a process for producing a cyclodextrin glycosyl transferase (CGTase) in B. licheniformis, in which process a suitable strain of B. licheniformis transformed with a DNA sequence which includes a gene coding for a CGTase, which DNA sequence is preceded by a promoter sequence capable of effecting transcription of said gene, is cultured under suitable conditions for the production of the CGTase, and the CGTase is recovered from the culture.

Although it has previously been suggested to clone the gene coding for debranching enzyme from Thermoanaerobium brockii into B_z_ subtilis (cf. R.D.Coleman et al. , J. Bacteriol. 169(9) , 1987, pp. 4302-4307) , and the CGTase gene from Thermoanaerobacter sp. into JL. subtilis (cf. WO 91/09129) , the cloning of the Thermoanaerobacter sp. CGTase gene, or other genes derived from anaerobic or thermophilic microorganisms, in Bacillus licheniformis appears to be novel. The cloning of heterologous genes into EL. licheniformis is generally more complicated that into, for instance, B^. subtilis. as most strains of B_j_ licheniformis cannot, at least at present, be transformed by being made competent and must be transformed by, for instance, protoplast formation resulting in a lower transformation frequency. However, B^. licheniformis is an advantageous microorganism to use for the production of recombinant enzymes as at least some strains of B. licheniformis produce large amounts of enzyme protein. It is therefore possible to obtain a higher yield of CGTase and other enzymes derived from anaerobic organisms in B____ licheniformis than in for instance B____ subtilis.

DETAILED DISCLOSURE OF THE INVENTION

According to the invention, the DNA sequence including the anaerobic and/or thermophilic gene should be operably connected to a suitable promoter sequence. The promoter may be any DNA sequence which shows transcriptional activity in B. licheniformis and may be derived from a gene encoding a protein homologous or heterologous to J _. licheniformis. Examples of suitable promoters are derived from the gene coding for B. stearothermophilus maltogenic amylase (amyM) , B^ licheniformis α-amylase (amyL) , B__s_ amyloliαuefaciens α-amylase (amyQ) , B. subtilis alcaline protease, or the B^ pumilus xylosidase promoter or the hybrid SPOl/lac promoter (D.G. Yansura and D.J. Henner, Proc. Natl. Acad. Sci. USA 81, 1984, pp. 439-443).

A particularly preferred promoter for use in the present process is a JL. licheniformis α-amylase promoter variant included in the following DNA sequence

GCATGCGTCC TTCTTTGTGC TTGGAAGCAG AGCCCAATAT TATCCCGAAA CGATAAAACG GATGCTGAAG GAAGGAAACG AAGTCGGCAA CCATTCCTGG GACCCATCCG TTATTGACAA GGCTGTCAAA CGAAAAAGCG TATCAGGAGA TTAACGACAC GCAAGAAATG ATCGAAAAAA TCAGCGGACA CCTGCCTGTA CACTTGCGTC CTCCATACGG CGGGATCAAT GATTCCGTCC GCTCGCTTTC CAATCTGAAG GTTTCATTGT GGGATGTTGA TCCGGAAGAT TGGAAGTACA AAAATAAGCA AAAGATTGTC AATCATGTCA TGAGCCATGC GGGAGACGGA AAAATCGTCT TAATGCACGA TATTTATGCA ACGTTCGCAG ATGCTGCTGA AGAGATTATT AAAAAGCTGA AAGCAAAAGG CTATCAATTG GTAACTGTAT CTCAGCTTGA AGAAGTGAAG AAGCAGAGAG GCTATTGAAT AAATGAGTAG AAAGCGCCAT ATCGGCGCTT TTCTTTTGGA AGAAAATATA GGGAAAATGG TACTTGTTAA AAATTCGGAA TATTTATACA ATATCATATG TTACACATTG AAAGGGGAGG AGAATC (SEQ ID#1) or a functional derivative thereof.

The thermophilic donor microorganism may be a strain of Archaebacterium and, more specifically, the gene derived from the thermophilic microorganism may therefore suitably be one encoding a Pyrococcus sp. pullulanase or α-amylase. The Pyrococcus sp. pullulanase and α-amylase may, for instance, be the one described in PCT/DK91/00219 and WO 90/11352, respectively. The anaerobic donor microorganism may be one which is also thermophilic, and the gene derived from the thermophilic and anaerobic microorganismmay therefore suitably be one encoding Thermoanaerobacter sp. or Thermoanaerobium sp. cyclodextrin glycosyl transferase, Thermotoga sp. glucose isomerase.

According to the invention, the DNA sequence including the gene derived from an anaerobic and/or thermophilic microorganism is present on an autonomously replicated expression vector. The vector further comprises a DNA sequence enabling the vector to replicate in the host cell. Examples of such sequences are the origins of replication of plasmids pUC19 (C. Yanisch-Perron et al., Gene 33, 1985, pp. 103-119), pACYC177 (A.C.Y. Chang and

S.N. Cohen, J. Bacteriol. 134. 1978, pp. 1141-1156), pUBHO

(Gryczan et al. 1978) or pIJ702 (E. Katz et al., J. Gen.

Microbiol. 129, 1983, pp. 2703-2714). The vector may also comprise a selectable marker, e.g. a gene whose product confers antibiotic resistance such as ampcillin, chloramphenicol, kanamycin or tetracyclin resistance, or the dal genes from B. subtilis or B^ licheniformis (B. Diderichsen, 1986) . The procedures used to ligate the DNA sequence coding for the gene from the anaerobic and/or thermophilic microorganism, promoter and origin of replication are well known to persons skilled in the art (cf. , for instance, Sambrook et al.. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY, 1989) .

Alternatively, the DNA sequence including the gene derived from an anaerobic and/or thermophilic microorganism may be present on the chromosome of the B^ licheniformis host cell. This is often an advantage as the DNA sequence is more likely to be stably maintained in the host cell. Integration of the DNA sequence into the host chromosome may be performed according to conventional methods, e.g. by homologous recombination. In one embodiment, said DNA sequence may be present in two or more copies on the chromosome of the B_^_ licheniformis host cell.

In a currently preferred embodiment of the present process, said DNA sequence is present on the chromosome of the B__j_ licheniformis host cell at the site of the B_j_ licheniformis α- amylase gene, and is expressed by means of the expression signals of the B____ licheniformis α-amylase, including the amyL promoter, in particular the amyL promoter variant described above, and the amylase signal peptide.

It is preferred that the B_j_ licheniformis host cell is one which is protease and/or amylase deficient as, generally speaking, it is an advantage that as few proteins as possible are present in the culture medium, thus facilitating the purification of the protein of interest. An expressed protease might also degrade at least part of the gene product of interest, and an expressed amylase (insofar as the gene product of interest is a starch-degrading enzyme such as CGTase) might not be tolerated in the final product and might make the subsequent purification of the product particularly difficult, either case resulting in a decreased yield of the product of interest. Protease and/or amylase deficiency may for instance be obtained by deletions or insertions in the genes encoding the protease or amylase, e.g. by introducing the DNA sequence encoding a CGTase into the host chromosome at the site of the α-amylase gene, as indicated above.

In the process of the invention, the product of the expressed gene is preferably recovered from the culture. Recovery of the product may be done by conventional procedures including separating the cells from the medium by centrifugation or filtration, precipitating the proteinaceous components of the supernatant or filtrate by means of a salt., e.g. ammonium sulphate, followed, if necessary, by a variety of chromatographic procedures, e.g. ion exchange chromatography, affinity chromatography, or the like.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is further described in the following example with reference to the appended drawings, in which the following abbreviations are used:

"pBR322" indicates pBR322-derived DNA;

"+ori pUBHO" indicates the plus origin of replication of pUBllO;

"rep" indicates the rep gene of pUBllO;

"cat" indicates the chloramphenicol resistance gene of pC194; "cgtA" indicates the Thermoanaerobacter CGTase gene;

"PamyM" indicates the promoter of the B^. sterothermophilus maltogenic amylase gene (Diderichsen and Christiansen, 1988) ;

"bla" indicates the ampicillin resistance gene of pBR322;

"PKK233-2" indicates pKK233-2 derived DNA; "PamyL" indicates the promoter of the B_-_ licheniformis a- a ylase gene;

"Pa yQ" indicates the promoter of the B . amyloliquefaciens α- amylase gene?

"amyL-cgtA" indicates the fusion gene comprising the signal peptide coding part of the B^ licheniformis α-amylase gene and the part of the Thermoanaerobacter CGTase gene coding for the mature enzyme;

"erm" indicates the erythromycin resistance gene of pE194;

"ori pE194" indicates the plus origin of replication and rep gene containing region of pE194;

"*amyL" indicated a DNA fragment spanning the 3'-end of the B. licheniformis α-amylase gene; "dal" indicates the gene coding for D,L-alanine racemase of B_. subtilis; and

"dfs" indicates a sequence immediately 3¹ of the dal gene.

is a restriction map of plasmid pNV601; is a restriction map of plasmid pPL1878; is a restriction map of plasmid pPL1419; is a restriction map of plasmid pPL1489; is a restriction map of plasmid pPL1540; is a restriction map of plasmid pDN3000; is a restriction map of plasmid pPL1759; is a restriction map of plasmid pPL1892; Fig. 9 is a restriction map of plasmid pPL1796; Fig. 10 is a restriction map of plasmid pBB37; Fig. 11 is a restriction map of plasmid pPL1385; Fig. 12 is a restriction map of plasmid pPL1893; Fig. 13 is a restriction map of plasmid pSJllll; Fig. 14 is a restriction map of plasmid pDN3060; Fig. 15 is a restriction map of plasmid pSJ1277; Fig. 16 is a restriction map of plasmid pSJ994; Fig. 17 is a restriction map of plasmid pSJ1283; Fig. 18 is a restriction map of plasmid pSJ1342; Fig. 19 is a restriction map of plasmid pSJ1359; Fig. 20 is a restriction map of plasmid pPL1483; Fig. 21 is a restriction map of plasmid pPL1487; Fig. 22 is a restriction map of plasmid pSJ932; Fig. 23 is a restriction map of plasmid pSJ948; Fig. 24 is a restriction map of plasmid pSJ980; Fig. 25 is a restriction map of plasmid pSJ1391; Fig. 26 is a schematic presentation of the exchange, by homologous recombination, between the chromosomal α-amylase gene and the amyL-cgtA fusion gene carried on plasmid pSJ1391; Fig. 27 is a schematic presentation of the .in vivo recombination between the 5¹ ends of the mature parts of cgtA; Fig. 28 is a restriction map of plasmid pDN1316; Fig. 29 is a restriction map of plasmid pDN3020; Fig. 30 is a restriction map of plasmid pSJ1446; and Fig. 31 is a restriction map of plasmid pSJ1448.

The invention is further illustrated in the following examples which are not in any way intended to limit the scope of the invention as claimed.

EXAMPLE

General Methods

The experimental techniques used to construct the plasmids were standard techniques within the field of recombinant DNA technology, cf. T. Maniatis et al.. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, New York, 1982.

Restriction endonucleases were purchased from New England Biolabs and Boehringer Mannheim and used as recommended by the manufacturers. T4 DNA ligase was purchased from New England Biolabs and used as recommended by the manufacturer.

Preparation of vector DNA from all strains was conducted by the method described by Kieser, 1984.

Transformation of E. coli:

Cells of E. coli were made competent and transformed as described by Mandel and Higa, 1970.

Transformation of B. subtilis:

Competent cells were prepared and transformed as described by

Yasbin et al., 1975.

Transformation of B. licheniformis: Plasmids were introduced into B. licheniformis by polyethylene glycol-mediated protoplast transformation as described by Akamatzu, 1984. CGTase-producing colonies of either L_. coli, B_-_ subtilis or B____ licheniformis were identified by plating transformants on LB agar plates supplemented with 1% soluble starch. After incubation at either 37°C or 30"C overnight, plates were 5 stained by iodine vapour to show hydrolysis zones produced by the action of the CGTase on the starch.

Media

BPX:

10

15 LB agar:

Adjusted to pH 7.5 with NaOH

201. Cloning of a Thermoanaerobacter sp. CGTase gene into Bacillus subtilis.

The construction of the E. coli plasmid pNV601 (Fig. 1) , carrying the Thermoanaerobacter sp. ATCC 53627 CGTase gene referred to in the following as cgtA, is disclosed in WO 2589/03421. The B. subtilis plasmid pPL1878 (Fig. 2), containing the cgtA gene, is disclosed in WO 91/09129. It was constructed as follows:

pNV601 was digested partially with Sau3A, then religated and transformed into E. coli SCSI (frozen competent cells purchased

30 from Stratagene, Ja Jolla, California) , selecting for ampicillin resistance (200 μg/ml) . One CGTase positive colony was PL1419, containing pPL14l9 (Fig. 3). Plasmid pPL1419 was partially digested with Sau3A, and fragments ligated to Bglll digested pPL1489 (Fig. 4) . One CGTase positive, ampicillin 5 resistant (200 μg/ml) E. coli SCSI transformant contained pPL1540 (Fig. 5) . pPL1489 was derived from plasmid pKK233-2 (purchased from Pharmacia LKB Biotechnology) by insertion of a synthetic DNA linker between the PstI and Hindlll sites in pKK233-2. This linker was the Pstl-Hindlll fragment from

10 pDN3000 (Fig. 6; WO 91/09129, Diderichsen et al., 1990). pPL1540 was digested with Haell and SphI, and the 2.4 kb fragment containing the cgtA gene was inserted into Haell + SphI digested plasmid pDN1380 (Diderichsen and Christiansen, 1988) . A CGTase positive, chloramphenicol resistant (6 μg/ml)

15 transformant of B. subtilis DN1885 (Diderichsen et al. , 1990) contained pPL1878.

2. Construction of an α-amylase/CGTase fusion gene.

Cloning of the Bacillus licheniformis α-amylase gene, amyL, resulting in plasmid pDN1981, is described by Jørgensen et al., 201990.

In plasmid pPL1759 (Fig. 7) , the Pstl-Hindlll fragment of pDN1981 has been replaced by the Pstl-Hindlll multilinker fragment from pDN3000 (Fig. 6) . It has retained the amyL promoter and most of the signal peptide coding sequence.

5 Plasmid pPL1892 (Fig. 8) was constructed by insertion of the cgtA gene excised from pPL1878 on a 2.4 kb Sall-NotI fragment into Sail + NotI digested pPL1759, and transformation of DN1885 to kanamycin resistance (10 μg/ml) .

Plasmid pPL1796 (Fig. 9) was constructed by insertion of a 0.5 0 kb SacI-EcoRV fragment from pBB37 (Fig. 10; Jørgensen, P. et al., 1991) into SacI + Smal digested pPL1385 (Fig. 11; Diderichsen et al., 1990), and transformation of DN1885 to chloramphenicol resistance (6 μg/ml) .

Plasmid pPL1893 (Fig. 12) was constructed by insertion of the CGTase gene excised from pPL1878 on a 2.4 kb BamHI-NotI fragment into BamHI + NotI digested pPL1796, and transformation of DN1885 to chloramphenicol resistance (6 μg/ml) .

The in vivo genetic engineering technique (Jørgensen et al. , 1990) , by which two DNA sequences contained on the same plasmid and sharing a homologous region can be fused together by recom- bination between the homologous regions in vivo (see Fig. 29) was used to construct a fusion between the amyL and the cgtA genes, in which the cgtA signal peptide coding sequence had been precisely replaced by the signal peptide coding sequence of the amyL gene.

To this end, the following oligonucleotide linker was syn¬ thesized and ligated into Sail digested pUC19 (Yanish-Perron et al., 1985), giving pSJllll (Fig. 13) upon transformation of E. coli SJ2 (Diderichsen et al., 1990) and selection for ampicillin resistance (200 μg/ml) :

3¹ end of amyL signal peptide coding region Sail Bell PstI

5• - TCGACTGATCACTTGCTGCCTCATTCTGCAGCAGCGGCG- 3' - GACTAGTGAACGACGGAGTAAGACGTCGTCGCCGC-

5' end of cgtA mature protein coding region

Xbal Sail GCACCGGATACTTCAGTTTCTCTAGAG - 3¹ CGTGGCCTATGAAGTCAAAGAGATCTCAGCT - 5' (SEQ ID#3) The pC194 (Horinouchi and Weisblum, 1982) derived chloramphenicol resistance gene, cat, was excised from pDN3060 (Fig. 14; WO 91/09129) as a 1.1 kb BamHI-Bglll fragment and inserted into Bell digested pSJllll, giving pSJ1277 (Fig. 15) upon transformation of E. coli SJ 6 (Diderichsen et al., 1990) and selection for ampicillin (200 μg/ml) and chloramphenicol (6 μg/ml) resistance.

pSJ994 (Fig. 16) was constructed by ligation of the 0.6 kb Notl-Ncol fragment from pPL1893 to the 5.4 kb Notl-Ncol fragment from pPL1892, and transformation into B. subtilis DN1885, selecting for kanamycin resistance (10 μg/ml) .

pSJ1283 (Fig. 17) was constructed by ligation of the 1.1 kb Sail fragment from pSJ1277 to Sail digested pSJ994, and transformation into DN1885, selecting for kanamycin (10 μg/ml) and chloramphenicol (6 μg/ml) resistance.

pSJ1342 (Fig. 18) was constructed by deletion of the 1.1 kb PstI fragment from pSJ1283, and transformation into DN1885, selecting for kanamycin resistance (10 μg/ml) .

pSJ1359 (Fig. 19) was constructed by the actual in vivo recom- bination from pSJ13 2. There is homology between the start of the mature part of the CGTase gene and part of the synthetic oligonucleotide extending between PstI and Sail on pSJ1342. If the plasmid undergoes a recombination event between these two homologous regions, the unique sites for Xbal, Sail and BamHI will be deleted.

A batch of pSJ1342 prepared from host strain DN1885 was thoroughly digested with BamHI, Xbal and Sail, and the digested plasmid was directly (i.e. without ligation) transformed into competent cells of DN1885, selecting for kanamycin resistance (10 μg/ml) . This procedure strongly enriches for recombined plasmids, as linearized plasmid monomers are unable to transform B. subtilis competent cells (Mottes et al., 1979). Recombined plasmids would not be cleaved by the restriction enzymes, and thus exist as a mixture of monomeric and oligomeric forms well able to transform competent B. subtilis 5 cells. One transformant thus obtained contained pSJ1359. This plasmid contains the origin of replication of pUBllO (Lacey and Chopra, 1974, Gryczan et al., 1978, McKenzie et al., 1986), the pUBllO rep protein gene, the kanamycin resistance gene, and the B. licheniformis α-amylase (amyL) promoter and signal peptide 10 coding region perfectly fused to the DNA encoding the mature part of the CGTase from Thermoanaerobacter sp. ATCC 53627.

3. Construction of a chromosomal integration vector.

A 1.4 kb BamHI fragment containing the pUBllO kanamycin resistance gene (kan) was excised from plasmid pDN2904 (WO 1591/09129) , ligated to Bglll digested pDN3000 (Fig. 6) , transformed into E. coli SCSI selecting ampicillin resistance (100 μg/ml) , and pPL1483 (Fig. 20) was recovered from one such transformant.

This plasmid was then combined with a Bacillus vector

20 temperature sensitive for replication, plasmid pE194

(Horinouchi and Weisblum, 1982b) . pPL1483 was digested with

AccI, pE194 digested with Clal, the two linearized plasmids mixed, ligated, and transformed into B. subtilis DN1885 selecting kanamycin resistance (10 μg/ml) at 30 °C. One such 5 transformant contained pPL1487 (Fig. 21) .

A 3*-terminal fragment of the amyL gene was excised from plasmid pDN1528 (Jørgensen, S. et al., 1991) as a 0.7 kb Sall- Hindlll fragment, ligated to Sall+Hindlll digested pUC19, and transformed to E. coli SJ2, selecting for ampicillin resistance 0 (200 μg/ml). One such transformant contained pSJ932 (Fig. 22). Plasmid pSJ948 (Fig. 23) was obtained by insertion of a Bglll linker into Hindu digested pSJ932, once more selecting for ampicillin resistance (200 μg/ml) upon transformation of SJ2.

pSJ980 (Fig. 24) was constructed by ligation of the 5.1 kb Hindlll fragment of pPL1487 to Hindlll digested pSJ948, selecting for kanamycin resistance (10 μg/ml) in B. subtilis DN1885 at 30 °C.

Finally, pSJ1391 (Fig. 25) was constructed by ligation of the 4.0 kb Bglll fragment of pSJ1359 to the 5.6 kb Bglll fragment of pSJ980, selecting for kanamycin resistance (10 μg/ml) in DN1885 at 30 °C. This plasmid contains, on a vector temperature- sensitive for replication and conferring resistance to kanamycin and erythromycin, the promoter and upstream region (about 0.4 kb) from the B. licheniformis α-amylase gene (amyL) , the α-amylase/CGTase fusion gene (amyL-cgtA) , and then about 0.7 kb from the 3'-region of the α-amylase gene ('amyL) .

4. Transfer of the fusion gene to B. licheniformis and integration in the chromosome.

An α-amylase producing strain of B. licheniformis was transformed with pSJ1391 by the protoplast transformation pro¬ cedure (Akamatzu, 1984) . One regenerating, kanamycin resistant colony was isolated, and was found to produce both α-amylase and CGTase. Production of the two enzymes can be easily distinguished by separating proteins in the culture supernatant from shake flask cultures in BPX medium (WO 91/09129) on isoelectric focusing gels (e.g. using the Pharmacia Phast sys¬ tem) , followed by overlayering with an agarose gel containing 1 % soluble starch and subsequent staining by iodine vapour. The CGTase activity was detected at pi 4.5, the α-amylase activity at pi 8. When this transformant was analyzed for its plasmid content, it turned out that a recombination event between the incoming plasmid and the chromosome had taken place: A double recombination had exchanged the chromosomal α-amylase (amyL) gene and the plasmid borne amyL-cgtA fusion gene, so that the plasmid isolated carried the amyL gene (B. subtilis DN1885 transformed with this plasmid produced α-amylase) whereas the amyL-cgtA fusion gene now resided on the chromosome (Fig. 26) .

By propagation in TY medium (WO 91/09129) without kanamycin, strains were isolated that had spontaneously lost their plasmid (SJ1599, SJ1603-1607) .

The original B. licheniformis transformant was also subjected to experimental conditions to ensure chromosomal integration and subsequent excision of the plasmid, in order to promote recombination events. The transformant was plated on LB agar (WO 91/09129) with 10 μg/ml kanamycin at 50 °C, individual colonies restreaked a few times at 50 °C, and each then grown in successive overnight TY cultures at 30 °C without kanamycin to permit plasmid excision and loss. Kana^s isolates from each original 50 °C colony were incubated in BPX shake flasks and production of either α-amylase or CGTase determined by analysis on isoelectric focusing gels as above. The plasmid free strains analyzed all produced either CGTase or α-amylase. CGTase producing isolates are e.g. SJ1561-62, 1580-83, 1586-91 and 1595.

One strain, named SJ1608, appeared to produce CGTase in larger amounts than the others.

Southern blot analysis of strains SJ1561, 1562, 1599, 1606 and 1608 confirmed that these strains have the chromosomal amyL gene replaced by the amyL-cgtA gene.

The following results were obtained by quantitation of the CGTase activity produced on incubation in BPX shake flasks for 6 days at 37 °C (results from several experiments; the variation within each group of strains was mainly due to the use of different batches of shake flasks) :

Strain CGTase activity,

5 arbitrary units

SJ1561-62, 1580-83, 1586-91, 1595, 1599, 1603-07 1 - 7.5

SJ1608 200 - 275

105. Promoter analysis.

We have investigated whether the large difference in CGTase production between strain SJ1608 and the other strains containing the amyL-cgtA gene was due to differences in the amyL promoter responsible for the CGTase expression.

5 The amyL promoter sequence of the B. licheniformis host strain is shown in SEQ ID#2.

The promoter region from a number of the CGTase producing B. licheniformis strains was amplified from chromosomal DNA by the PCR technique (Saiki et al., 1988) , using as primers one oligo- 0 nucleotide corresponding to pos. 204-233 reading downstream through the amyL promoter, and another oligonucleotide corre¬ sponding in sequence to the 5'-end of the DNA encoding the mature CGTase and reading upstream. The sequence of this second oligonucleotide was 5'-CCTGTTGGATTATTACTGGG-3' (SEQ ID#4) .

5 The amplified DNA fragment from each strain was excised from an agarose gel and directly sequenced, using as sequencing primers in the dideoxy method (Sanger et al., 1977) the same oligonucleotides that were used for PCR amplification. The results of the sequence analysis reveal that one or both of two point mutations in the promoter region are responsible for the large difference in CGTase production observed.

Strains SJ1599 and 1603-06, all low-yielding, have the promoter 5 sequence shown in SEQ ID#2. However, the high-yielding strain SJ1608 contains the promoter sequence shown in SEQ ID#1.

The differences occur at pos. 553, where SJ1608 contains a C instead of a T, and at pos. 593, where SJ1608 contains a A instead of a T.

10 The sequence of the amyL promoter present of pSJ1359 and pSJ1391 was determined using the PCR amplification and sequencing procedure described above. This showed that both plasmids contain the promoter sequence shown in SEQ ID#1, i.e. identical to the promoter sequence of SJ1608.

156. Integration of the amyL-cgtA fusion σene in the B. subtilis chromosome

Strains containing one copy of the amyL-cgtA fusion gene in the dal region of _________ subtilis chromosome were constructed essentially as described in B. Diderichsen, 1986. pDN1316 (Fig. 2028) is identical to pDN1313 (B. Diderichsen, 1986) except for the orientation of the multilinker.

pDN3020 (Fig. 29) is a derivative of pDN1316 constructed by inserting a synthetic SphI site containing oligonucleotide linker into the EcoRI site of plasmid pDN1380 (Diderichsen and

25 Christiansen, 1988), resulting in plasmid pDN1620. The promoter region of a maltogenic amylase from B_-_ stearothermophilus (Pa yM) present on pDN1620 was then transferred to Sphl-BamHI digested pUC19 on an approximately 200 bp BamHI-SphI fragment, resulting in plasmid pDN2977. The promoter region was excised

30 from pDN2977 on an approximately 200 bp Bglll-SacI fragment which was inserted in the polylinker region of pDN1316, thereby generating plasmid pDN3020.

Strain DN1686 is a Spo^" derivative of DN1280 which contains a chromosomal deletion in the dal gene (Diderichsen, 1986) . DN1686 was derived from DN1280 by traditional mutagenesis procedures and was used as the host in the following experiment.

The amyL-cgtA fusion gene was excised from pSJ1360 (identical to pSJ1359 shown in Fig. 19) as a 4 kb Bglll fragment and ligated to BamHI digested pDN3020, resulting in pSJ1446 (Fig. 30) and pSJ1448 (Fig. 31) on transformation of DN1686 to chloramphenicol resistance (6 μg/ml) .

Integrant strains SJ1454 and SJ1455 were subsequently isolated by transformation of DN1686 with pSJ1446 and pSJ1448, respectively, and isolation of transformants that were CGTase- producing, but chloramphenicol sensitive. These strains were incubated in BPX shake flasks at 37°C for 6 days, and the CGTase activity was measured (in arbitrary unit, as in example 4).

Strain CGTase activity, arbitrary units

SJ1454 21

SJ1455 17

Comparison of the results obtained in Examples 4 and 6 demonstrates that B_j_ licheniformis is a significantly better host strain for the production of CGTase than B^ subtilis. REFERENCES.

Akamatzu, T. , Sekiguchi, J. (1984). An improved method of protoplast regeneration for Bacillus species and its application to protoplast fusion and transformation. Agric. Biol. Chem. , 48, 651-655.

Diderichsen, B. , Christiansen, L. (1988). Cloning of a maltogenic alpha-amylase from a Bacillus stearothermophilus. FEMS Microbiology Letters, 56, 53-60.

Jørgensen, P. L. , Hansen, C. K. , Poulsen, G. B. , Diderichsen, B. (1990) . In vivo genetic engineering: homologous recombination as a tool for plasmid construction. Gene 96, 37- 41.

Diderichsen, B. In: Bacillus Molecular Genetics and Biotechnology Applications. A.T. Ganesan and J.A. Hoch, eds., Academic Press, 1986, pp. 35-46.

Diderichsen, B. , Wedsted, U. , Hedegaard, L. , Jensen, B. R. , Sjøholm, C. (1990) . Cloning of aldB, which encodes a- acetolactate decarboxylase, an exoenzyme from Bacillus brevis. Journal of Bacteriology, 172, 4315-4321.

Jørgensen, P. L. , Poulsen, G. B. , Diderichsen, B. (1991). Cloning of a chromosomal α-amylase gene from Bacillus stearothermophilus. FEMS Microbiology Letters, 77, 271-276.

Jørgensen, S., Skov, K. W. , Diderichsen, B. (1991). Cloning, sequence, and expression of a lipase gene from Pseudomonas cepacia: Lipase production in heterologous hosts requires two Pseudomonas genes. Journal of Bacteriology, 173, 559-567. Horinouchi, S., Weisblum, B. (1982a). Nucleotide sequence and functional map of pC194, a plasmid that specifies chloramphenicol resistance. J. Bacteriol., 150, 815-825.

Horinouchi, S., Weisblum, B. (1982b). Nucleotide sequence and 5 functional map of pE194, a plasmid that specifies inducible resistance to macrolide, lincosamide, and streptogramin type B antibiotics. J. Bacteriol. 150, 804-814.

Kieser, T. (1984) . Factors affecting the isolation of CCC DNA from Streptomvces lividans and Escherichia coli. Plasmid 12, 1019-36.

Mandel, A., Higa, A. (1970). J. Mol. Biol. 53, 159-162.

Mottes, M., Grandi, G. , Sgaramella, V., Canosi, U. , Morelli, G., Trautner, T. A. (1979). Different specific activities af monomeric and oligomeric forms of plasmid DNA in transformation 15 of Bacillus subtilis and Escherichia coli. Mol. Gen. Genet., 174, 281-286.

Lacey, R. , Chopra, I. (1974). Genetic studies of a mul iresistant strain of Staphylococcus aureus. J. Med. Microbiol., 7, 285-297.

20 McKenzie , T., Hoshino, T., Tanaka, T., Sueoka, N. (1986). The nucleotide sequence of pUBllO: Some salient features in relation to replication and its regulation. Plasmid 15, 93-103.

Sanger, F., Nicklen, S., Coulson, A. R. (1977). DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA, 2574, 5463-5467.

Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi, R., Horn, G. T. , Mullis, K. B. , Erlich, H. A. (1988). Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239, 487-491. Gryczan, T. , Contente, S., Dubnau, D. (1978). Characterization of Staphylococcus aureus plasmids introduced by transformation into Bacillus subtilis. J. Bacteriol., 134, 318-329.

Yanish-Perron, C. , Vieira, J. , Messing, J. (1985). Improved Ml3 phage cloning vectors and host strains: nucleotide sequences of the M13 mplδ and pUC19 vectors. Gene 33, 103-119.

Yasbin, R. E., Williams, G. A., Young, F. E. (1975). J. Bacteriol. 121, 296-304.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: NOVO NORDISK A/S

(B) STREET: Novo Alle

(C) CITY: Bagsvaerd

(D) COUNTRY: Denmark

(E) POSTAL CODE (ZIP) : DK-2880

(ii) TITLE OF INVENTION: A Process for Expressing Anaerobic Genes in Bacillus licheniformis

(iii) NUMBER OF SEQUENCES: 4

(iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Novo Nordisk A/S

(B) STREET: Novo Alle

(C) CITY: Bagsvaerd

(D) COUNTRY: Denmark

(E) ZIP: 2880 (V) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version #1.25 (EPO)

(vi) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: PCT/DK91/003 4

(B) FILING DATE: 14-NOV-1991

(vii) ATTORNEY/AGENT INFORMATION: (A) NAME: Thalsø-Madsen, Birgit

(B) REFERENCE/DOCKET NUMBER: 3653.204-WO

(viii) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: +45 4444 8888

(B) TELEFAX: +45 4449 3256 (2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 616 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY : linear

( ii) MOLECULE TYPE : DNA (genomic)

(vi) ORIGINAL SOURCE :

(A) ORGANISM: Bacillus licheniformis

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

GCftTGCGTOC TTCITIGΓGC TTGGAAGCAG AGOCCAAIAT TAΓOOOGAAA αSATAAAAOG 60

GATGCTGAAG GAAGGAAACG AACTOGGCAA CCMTCC GG GAOCCATOOG TTA3TGACAA 120

GGCTGICAAA OGAAAAAGOG TATCAGGAGA TTAACGACAC GCAAGAAATG ATOGAAAAAA 180

TCAGOGGACA (XTGCCTGTA CACITGCGTC CTCCATAOGG OGGGATCAAT GATTCCGICC 240 GCTOGCITTC C&ATCTGAAG GTTTCATIGT GGGATGTTGA TOOGGAAGAT TGGAAGTACA 300

AAAATAAGCA AAAGAI CTC AATCATCTCA. TGAGOCATGC GGGAGAOGGA AAAATCGTCT 360

TAATGCAOGA TA3TTATGCA AOGTTOGCAG ATGCTGCTGA AGAGATTA T AAAAAGCTGA 420

AAGCAAAAGG CTATCAA1TG GTAAOPGTAT CTCAGCTIGA AGAAGIGAAG AAGCAGAGAG 480

GCEATTGAAT AAATGAGEAG AAAGOGCCAT ATOGGCGCTT TTCITTΓGGA AGAAAATAIA 540 GGGAAAATGG TACTTGTTAA AAATTOGGAA TATITAIACA ATATCATATG ITACACAITG 600

AAAGGGGAGG AGAATC 616 (2) D OEM πO EOR SEQ ID NO: 2 :

(i) SEQUENCE O.ARACTERISTICS:

(A) IENGTH: 616 base pairs (B) TYPE: nucleic acid

(C) STRANDEENESS: single

(D) TOPOIOGY: linear

(ii) MDIECUIE TYPE: DNA (gencmic)

(Vi) ORIGINAL SOURCE: (A) ORGANISM: Bacillus licheniformis

(B) STRAIN: SJ1608

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

GCATGOGTOC TTCITTGTGC TTGGAAGCAG AGOCCAAIAT TATCCOSAAA OGATAAAAOG 60

GATGCTGAAG GAAGGAAAOG AACTOGGCAA CXMTCCTGG GACOCATCOG TTATTGACAA 120 GGCTGICAAA OGAAAAAGOG TATCAGGAGA TTAACGACAC GCAAGAAATG ATOGAAAAAA 180

TCAGOGGACA CCTGCCIGIA CACπGOGTC CTOCAIAOSG OGGGATCAAT GATTCOGTCC 240 GCTCGCΓITC CAATCTGAAG GOTTCAITCT GGGATGΠEA TOOGGAAGAT TGGAAGΓACA 300

ASAATAAGCA AAAGATTCRC AATC_ATSTCA TGAGOCATGC GGGAGAOGGA AAAATOGTCT 360

TAATGCAOGA TAM-AΪGCA AOSTTOGCAG ATGCTGCTGA. AGAGATTATT AAAAAGCTGA. 420

AAGCAAAAGG CTAKAATTG GTAACTGEAT CTCBGCITGA AGAAGTGAAG AAGCAGAGAG 480 GCTA3TGAAT AAATGAGEAG AAAGOGCCAT ATOGGOGCIT TTCTTITGGA. AGAAAATATA 540

GGGAAAATGG TAπTIGTTAA AAATTOGGAA TATTEAIACA. A3ATCAIATG TTTCACATTG 600

AAAGGGGAGG AGAATC 616

(2) INPOFMATION FOR SEQ ID NO: 3:

(i) SEQUENCE OJARACEERISTICS: (A) IENGTH: 66 base pairs

(B) TYPE: nucleic acid

(C) STRANEEENESS: single

(D) TOPOIOGY: linear

(ii) M3IECULE TΪPE: CENA (vi) ORIGINAL SOURCE:

(A) ORGANISM: synthetic

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: TOGACTGATC ACTTGCTGCC TCATTCTGCA GCAGOGGOGG CAOOGGATAC TTCaGTTTCT 60

CTAGAG 66 (2) INFOEMAnCN FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) IENSTH: 20 base pairs

(B) TΪEE: nucleic acid

(C) SttRANDEENESS: single (D) TOPOIOGY: linear

(ii) MOIECUIE TYPE: cENA

(vi) ORIGINAL SOURCE:

(A) ORGANISM: synthetic

(xi) SEQUENCE EESCRHTION: SEQ ID NO: 4: CCTGETGGAT TATTACTGGG 20

Claims

1. A process for expressing genes derived from anaerobic and/or thermophilic microorganisms in Bacillus licheniformis, in which process a suitable strain of B . licheniformis transformed with a DNA sequence which includes a gene derived from an anaerobic and/or thermophilic microorganism, which DNA sequence is preceded by a promoter sequence capable of effecting transcription of said gene, is cultured under suitable conditions to obtain gene expression.

2. A process according to claim 1, wherein the promoter is derived from the gene coding for B. stearothermophilus maltogenic amylase, B. licheniformis α-amylase, B. amyloliquefaciens α-amylase, B . subtilis alcaline protease, or the B. pumilus xylosidase promoter or the hybrid SPO1/lac promoter.

3. A process according to claim 2, wherein the promoter is a B. licheniformis α-amylase promoter variant included in the following DNA sequence

GCATGCGTCC TTCTTTGTGC TTGGAAGCAG AGCCCAATAT TATCCCGAAA CGATAAAACG GATGCTGAAG GAAGGAAACG AAGTCGGCAA CCATTCCTGG

GACCCATCCG TTATTGACAA GGCTGTCAAA CGAAAAAGCG TATCAGGAGA

TTAACGACAC GCAAGAAATG ATCGAAAAAA TCAGCGGACA CCTGCCTGTA

CACTTGCGTC CTCCATACGG CGGGATCAAT GATTCCGTCC GCTCGCTTTC

CAATCTGAAG GTTTCATTGT GGGATGTTGA TCCGGAAGAT TGGAAGTACA AAAATAAGCA AAAGATTGTC AATCATGTCA TGAGCCATGC GGGAGACGGA

AAAATCGTCT TAATGCACGA TATTTATGCA ACGTTCGCAG ATGCTGCTGA

AGAGATTATT AAAAAGCTGA AAGCAAAAGG CTATCAATTG GTAACTGTAT

CTCAGCTTGA AGAAGTGAAG AAGCAGAGAG GCTATTGAAT AAATGAGTAG

AAAGCGCCAT ATCGGCGCTT TTCTTTTGGA AGAAAATATA GGGAAAATGG TACTTGTTAA AAATTCGGAA TATTTATACA ATATCATATG TTACACATTG

AAAGGGGAGG AGAATC (SEQ ID#1) or a functional derivative thereof.

4. A process according to claim 1, wherein the thermophilic microorganism is a strain of Archaebacterium.

5. A process according to claim 4, wherein the the gene derived from the thermophilic microorganism is one encoding Pyrococcus sp. pullulanase or α-amylase.

6. A process according to claim 1, wherein the anaerobic microorganism is also thermophilic.

7. A process according to claim 6, wherein the gene derived from the thermophilic and anaerobic microorganism is one encoding Thermoanaerobacter sp. or Thermoanaerobium sp. cyclodextrin glycosyl transferase, or Thermotoga sp. glucose isomerase.

8. A process according to claim 1, wherein the DNA sequence including the gene derived from an anaerobic and/or thermophilic microorganism is present on an autonomously replicated expression vector.

9. A process according to claim 1, wherein the DNA sequence including the gene derived from an anaerobic and/or thermophilic microorganism is present on the chromosome of the B. licheniformis host cell.

10. A process according to claim 9, wherein said DNA sequence is present in two or more copies on the chromosome of the B. licheniformis host cell.

11. A process according to claim 9 or 10, wherein said DNA sequence is present on the chromosome of the B. licheniformis host cell at the site of the B. licheniformis α-amylase gene. and is expressed by means of the expression signals of the B. licheniformis α-amylase.

12. A process according to claim 1, wherein the product of the expressed gene is subsequently recovered from the culture.

13. A process according to any of claims 1-12, wherein the B. licheniformis host cell is protease and/or amylase deficient.

14. A process for producing a cyclodextrin glycosyl transferase (CGTase) in B. licheniformis. in which process a suitable strain of licheniformis transformed with a DNA sequence which includes a gene coding for a CGTase, which DNA sequence is preceded by a promoter sequence capable of effecting transcription of said gene, is cultured under suitable conditions for the production of the CGTase, and the CGTase is recovered from the culture.

15. A process according to claim 14, wherein the promoter is derived from the gene coding for B. stearothermophilus maltogenic amylase, B. licheniformis α-amylase, B. amyloliquefaciens BAN amylase, B. subtilis alcaline protease, or the B. pumilus xylosidase promoter or the hybrid SPO1/lac promoter.

16. A process according to claim 15, wherein the promoter is a B. licheniformis α-amylase promoter variant included in the following DNA sequence

GCATGCGTCC TTCTTTGTGC TTGGAAGCAG AGCCCAATAT TATCCCGAAA CGATAAAACG GATGCTGAAG GAAGGAAACG AAGTCGGCAA CCATTCCTGG GACCCATCCG TTATTGACAA GGCTGTCAAA CGAAAAAGCG TATCAGGAGA TTAACGACAC GCAAGAAATG ATCGAAAAAA TCAGCGGACA CCTGCCTGTA CACTTGCGTC CTCCATACGG CGGGATCAAT GATTCCGTCC GCTCGCTTTC CAATCTGAAG GTTTCATTGT GGGATGTTGA TCCGGAAGAT TGGAAGTACA AAAATAAGCA AAAGATTGTC AATCATGTCA TGAGCCATGC GGGAGACGGA AAAATCGTCT TAATGCACGA TATTTATGCA ACGTTCGCAG ATGCTGCTGA AGAGATTATT AAAAAGCTGA AAGCAAAAGG CTATCAATTG GTAACTGTAT

CTCAGCTTGA AGAAGTGAAG AAGCAGAGAG GCTATTGAAT AAATGAGTAG AAAGCGCCAT ATCGGCGCTT TTCTTTTGGA AGAAAATATA GGGAAAATGG TACTTGTTAA AAATTCGGAA TATTTATACA ATATCATATG TTACACATTG AAAGGGGAGG AGAATC (SEQ ID#1) or a functional derivative thereof.

17. A process according to any of claims 14-16, wherein gene encoding the CGTase is one derived from a Thermoanaerobacter sp., Thermoanaerobium sp., Bacillus macerans, Bacillus circulans, Bacillus stearothermophilus, Bacillus megaterium. Bacillus ohbensis, alkalophilic Bacillus sp., Micrococcus luteus, Micrococcus varians or Klebsiella pneumoniae.

18. A process according to claim 14, wherein the gene coding for CGTase is present on an autonomously replicated expression vector.

19. A process according to claim 14, wherein the gene coding for CGTase is present on the chromosome of the B. licheniformis host cell.

20. A process according to claim 19, wherein said gene is present in two or more copies on the chromosome of the B. licheniformis host cell.

21. A process according to claim 19 or 20, wherein said gene is present on the chromosome of the B. licheniformis host cell at the site of the B. licheniformis α-amylase gene, and is expressed by means of the expression signals of the B. licheniformis α-amylase.

22. A process according to any of claims 14-21, wherein the B. licheniformis host cell is protease and/or amylase deficient.