WO1999001562A1

WO1999001562A1 - Protein producing cell containing multiple copies of a desired gene and a screenable marker but no selection marker

Info

Publication number: WO1999001562A1
Application number: PCT/DK1998/000300
Authority: WO
Inventors: Steen Troels JØRGENSEN; Kim Brint Pedersen
Original assignee: Novo Nordisk AS
Current assignee: Novo Nordisk AS
Priority date: 1997-07-03
Filing date: 1998-07-02
Publication date: 1999-01-14
Anticipated expiration: 2000-01-03
Also published as: AU7908998A; EP1002109A1; CN1261918A; JP2002508669A

Abstract

A method of constructing a microbial multicopy protein production cell which does not comprise an inserted selection marker gene; a microbial multicopy protein production cell obtainable by said method; and a method of producing a protein of interest using such a microbial multicopy protein production cell.

Description

PROTEIN PRODUCING CELL CONTAINING MULTIPLE COPIES OF A DESIRED GENE AND A SCREENABLE MARKER BUT NO SELECTION MARKER

FIELD OF INVENTION

The present invention relates to a method of constructing a microbial multicopy protein production cell which does not comprise an inserted selection marker gene; a microbial multicopy protein production cell obtainable by said method; and a method of producing a protein of interest using such a microbial multicopy protein production cell.

BACKGROUND OF THE INVENTION

The art describes a number of techniques to make microbial strains which are capable of expressing high amount of a protein of interest. Such techniques are generally based on construction of multicopy strains, i.e. strains which comprise more than one copy of a gene encoding the protein of interest.

Such multicopy strains are generally constructed by the following strategy: 1) introducing a sequence into the chromosome of a microbial cell whereby the chromosome of the cell comprises the DNA structure A-P-M-A, in which A denotes homologous DNA sequences,

P denotes a DNA sequence encoding a protein of interest, M denotes a DNA sequence encoding a selection marker (e.g. an antibiotic resistance) ,

2) propagating said cell under increasing selective pressure for the selection marker:

3) identifying cells which have obtained increased resis- tance by homologous recombination at the homologous sequences A.

The constructed multicopy strains then comprise the structure A-P-M-A-P-M-A-P-M-A .

US 4959316, US 5695976, US 5733753 and WO 94/14968 describe such techniques for construction of multicopy strains, and reference is made thereto for further details. SUMMARY OF THE INVENTION

Use of selection markers, especially antibiotic resistance markers, is not preferred in cells used for industrial production of proteins among others due to environmental concerns. Accordingly, the problem to be solved by the present invention is to provide a new technique for construction of a multicopy cell without using any selection markers in the actual multicopy isolation procedure and thereby providing the possibility of obtaining a multicopy cell which does not comprise inserted selection marker genes, in particular not inserted antibiotic resistance marker genes.

The solution is based on that the present inventors have demonstrated that it is possible to construct a multicopy strain by using a screenable protein as a marker instead of the in the art known use of a selectable protein as a marker.

Accordingly, in an first aspect the present invention relates to a method of construction a microbial multicopy protein production cell which does not comprise an inserted selection marker gene, comprising a) introducing a sequence into the chromosome of a microbial cell whereby the chromosome of the cell comprises the DNA structure A-P-S-A, in which A denotes homologous DNA sequences,

P denotes a DNA sequence encoding a protein of interest, S denotes a DNA sequence encoding a screenable protein; b)propagating said cell; c) screening for a cell which produces increased amounts of said screenable protein; and d) recovering the cell identified in c) ; and optionally e) repeating steps b-d using a recovered cell from step d) as starting material.

The term "screenable protein" denotes a protein which is not essential for growth of the cell.. i.e. if it is removed from the cell, then said cell is still capable of growing at substantially the same growth rate, under similar growth conditions, as compared to when said screenable protein is com- prised within said cell. A suitable "screenable protein" may be a protein which is fluorescent under suitable exposure, such as Green Fluorescent protein (GFP) or variants thereof (see below for further details) . This is contrary to a protein herein termed "selectable protein" ; "selectable marker protein"; or "selection marker protein" denoting a protein which is "essential for growth of the cell" , i.e. if it is removed from the cell, then said cell will NOT be capable of growing at substantially the same growth rate, under similar growth conditions, as compared to when said "selectable protein" is comprised within said cell.

Such a "selectable marker protein" may typically be an antibiotic resistance protein. Accordingly, when a cell is grown in a medium comprising the corresponding antibiotic the amount of said antibiotic resistance protein within the cell may substantially affect the actual growth rate of the cell.

The term "multicopy protein production cell which does not comprise an inserted selection marker gene" denotes a cell which does not comprise a selection marker gene which has been in- serted into the cell during the process of making the multicopy protein production cell. See the "BACKGROUND" section above for discussion of processes known in the art leading to multicopy protein production cells comprising such inserted selection marker genes. The term "a gene" denotes a DNA sequence encoding a polypeptide with the specified activity, i.e. the term "selection marker gene" denotes a DNA sequence encoding a polypeptide having the selection marker activity.

The multicopy cell, identified according to the method of the invention, has obtained an increased number of the screenable protein "S" gene in the chromosome by spontaneous homologous recombination at the homologous sequences "A" .

Spontaneous homologous recombinations are generally a rather rare event. However by screening a suitable population of cells

(preferably more than 10⁵ individual cells) according to the method of the invention, the present inventors have demonstrated that it is possible to identify such a cell. The final constructed multicopy cell, according to the invention, comprises the structure A-P-S-A-P-S-A-P-S-A , and thereby comprises multicopies of the protein of interest "P" .

One of the advantages of such a multicopy cell produced ac- cording to a method of the invention, is it does not comprise an inserted selection marker gene "M" (see "BACKGROUND" section above, for description of in the art known methods, wherein the final constructed multicopy cell is characterized by comprising such inserted selection marker genes) . The presence of such genes, in particular antibiotic resistance marker genes, are undesirable, in particular from an environmental point and product approval point of view.

In a second aspect the present invention relates to a microbial multicopy protein production cell obtainable by any of the methods according to the invention, characterized by that said cell comprises multiple copies of a gene expressing a protein of interest ("P") and multiple copies of a gene expressing a screenable protein ("S") .

The term "multiple copies of a gene expressing a protein of interest ("P") " denotes that said cell comprises at least 2 copies of said gene expressing a protein of interest; more preferably that said cell comprises at least 4 copies of said gene expressing a protein of interest; and even more preferably that said cell comprises at least 7 copies of said gene expressing a protein of interest.

The term "multiple copies of a gene expressing a screenable protein ("S") " similarly denotes that said cell comprises at least 2 copies of said gene expressing a screenable protein; more preferably that said cell comprises at least 4 copies of said gene expressing a screenable protein; and even more preferably that said cell comprises at least 7 copies of said gene expressing a screenable protein.

In a third aspect the present invention relates to a process for production of at least one protein of interest in a mi- crobial cell, which method comprises: i) culturing a microbial multicopy protein production cell according to claim 9 under conditions permitting the production of the protein of interest; and ii) recovering said protein of interest from the resulting culture broth or the microbial multicopy protein production cell.

Embodiment(s) of the present invention is described below, by way of examples only.

DRAWINGS

Figures 1-9: The figures show plasmids used in working examples 1 and 2 herein to make a microbial multicopy protein production cell, according to the invention, by a method for constructing a microbial multicopy protein production cell, according to the invention. Consequently, reference is made to examples 1 and 2, for further description of said plasmids.

DETAILED DESCRIPTION OF THE INVENTION

Introducing a sequence into the chromosome of a microbial cell whereby the chromosome of the cell comprises the DNA structure A-P-S-A:

Introducing a sequence into the chromosome of a microbial cell whereby the chromosome of the cell comprises the DNA struc- ture A-P-S-A (step a) ) may be performed by a similar strategy known in the art to build in a DNA structure comprising the structure A-P-M-A into the chromosome of a microbial cell ("M" is a selection marker gene) .

Building up of such DNA structure into the chromosome of a microbial cell may be performed by introducing a vector comprising the structure A-P-S-A, or more preferably the structure A-P-S, into a microbial cell followed by a recombination of said structure into the chromosome of said microbial cell. Alternatively a DNA segment comprising a screenable protein "S" may be directed into a chromosome already comprising the structure A-P- A in order to make a DNA structure A-P-S-A on the chromosome in a similar manner. For a person skilled in the art it is routine work to select a particular suitable strategy to introduce such a A-P-S-A structure into the chromosome of a microbial cell.

WO 94/23073, US 5695976, US 5733753, and US 4959316 de- scribe such strategies in great details, and those references are incorporate herein for a detailed description to perform introduction of such DNA structures into the chromosome of a microbial cell.

Further, examples of suitable strategies are described in working examples herein (vide infra) .

Alternatively, the term "introducing a sequence into the chromosome of a microbial cell whereby the chromosome of the cell comprises the DNA structure A-P-S-A" may be termed "introducing a DNA construct comprising the structure A-P-S-A into the chromosome of a microbial cell".

Screening for a cell which produces increased amounts of the screenable protein:

In order to identify a multicopy cells according to the invention it is preferred to screen a large number of individual cells. Preferably (in step c) ) at least 10⁵ individual cells are screened, more preferably at least 10 individual cells are screened, and even more preferably are at least 10⁷ individual cells are screened. In an embodiment of the invention the screenable protein in step c) is a protein which is fluorescent under suitable exposure, and in particular said fluorescent protein is Green Fluorescent protein (GFP) or variants thereof.

Green Fluorescent protein (GFP) or variants thereof are described in the art and reference is made to Crameri et al. Nature Biotechnology 14:315-319 (1996); Cormack et al. GENE 173:33-38 (1996); and WO 97/11094 for further details. GFP is fluorescent under suitable light exposure.

An example of an alternative suitable screenable protein may be a beta-galactosidase, which may be detected with suitable fluorogenic enzyme substrates according to methods known in the art. The screening may be performed in any standard screening system for measuring amounts of a fluorescent protein.

Due to the high screening capacity and available commercial apparatus for performing the technique it is preferred that the screening in step c) is performed by use of the known technique of flow cytometry using a flow cytometer with cell sorting capability (Cormack et al. "FACS-optimized mutants of the green fluorescent protein (GFP)" GENE 173:33-38 (1996)). An example of such a flow cytometer is FACSCalibur from Becton Dickinson and Company.

Accordingly, in a further embodiment of the invention the screening in step c) is performed by using a flow cytometer with cell sorting capability, and screening for cells which have increased content of a fluorescent screenable protein, and in par- ticular wherein said fluorescent protein is Green Fluorescent protein (GFP) or variants thereof, and GFP or variants thereof is made fluorescent under suitable light exposure during the screening procedure.

In an even further embodiment of the invention the micro- bial cell of the invention is a bacterial cell, in particular wherein said bacterial cell is a cell of the genus Bacillus, in particular a cell of Bacillus subtilis, Bacillus lentus, Bacillus brevis, Bacillus stearothermophilus, Bacillus alkalophilus , Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus cir- culans, Bacillus lautus, Bacillus thuringiensis, Bacillus clausii or Bacillus licheniformis.

In an even further embodiment of the invention the protein of interest ("P" in step a)) is an enzyme, in particular a protease, a lipase, an amylase, a galactosidase, a pullanase, a cellulase, a glucose isomerase, a protein disulphide isomerase, a CGT'ase (cyclodextrin gluconotransferase) , a phytase, a glucose oxidase, a glucosyl transferase, a laccase, or a xylanase.

A microbial multicopy protein production cell obtainable by any of the methods according to the invention:

As described above said multicopy protein production cell is characterized by that it comprises multiple copies of a gene expressing a protein of interest ("P") and multiple copies of a gene expressing a screenable protein ("S") .

As further indicated above said multicopy protein production cell is further characterized by that it does not comprise 5 a selection marker gene which has been inserted into the cell during the process of making the multicopy protein production cell.

A process for production of at least one protein of interest in

10 a microbial cell:

The culturing medium used to culture the microbial multicopy protein production cell may be any conventional medium suitable for growing the cells in question. The expressed protein of interest may conveniently be secreted into the

15 culture medium and may be recovered therefrom by well-known procedures including separating the cells from the medium by centrifugation or filtration, precipitating proteinaceous components of the medium by means of a salt such as ammonium sulphate, followed by chromatographic procedures such as ion

20 exchange chromatography, affinity chromatography, or the like.

The invention is further illustrated by the following non- limiting examples.

Further, the disclosures in Danish patent application DK 25 0792/97, from which this application claims priority, and in the abstract accompanying this application are incorporated herein as reference.

MATERIALS AND METHODS

30

General molecular biology methods:

Unless otherwise mentioned the DNA manipulations and transformations were performed using standard methods of molecular biology (Sambrook et al. (1989) Molecular cloning: A 35 laboratory manual, Cold Spring Harbor lab., Cold Spring Harbor, NY; Ausubel, F. M. et al. (eds.) "Current protocols in Molecular Biology". John Wiley and Sons, 1995; Harwood, C. R. , and Cutting, S. M. (eds.) "Molecular Biological Methods for Bacillus". John Wiley and Sons, 1990).

Unless otherwise mentioned PCR manipulations were performed using standard protocols and PCR reaction parameters. See e.g. the text-book reference ("PCR A practical approach" IRL Press, (1991)).

Enzymes for DNA manipulations were used according to the specifications of the suppliers.

Enzymes for DNA manipulations

Unless otherwise mentioned all enzymes for DNA manipulations, such as e.g. restriction endonucleases, ligases etc., are obtained from New England Biolabs, Inc.

Flow cytometrv

Flow cytometry is carried out on a FACSCalibur flow cytometer from Becton Dickinson.

Flow cytometry on the FACSCalibur flow cytometer is carried out according to the guidelines of the supplier, e.g. as described in FACSCalibu System User's Guide, August 1996. Flow cytometric terminology is used as in said FACSCalibur™ Systems User's Guide.

Media (TY, BPX and LB agar) have been described in EP 0 506 780.

LBPSG agar is LB agar supplemented with phosphate (0.01 M K₃P0 ) , glucose (0.4 %) , and starch (0.5 %)

EXAMPLE 1 Construction of a bacterial strain harbouring an amplifiable cassette comprising a classical antibiotic resistance marker gene, and a screenable gene encoding green fluorescent protein

Plasmid constructions:

A) pSJ2773

Plasmid pSJ2059 and its use as a tool to generate amplifications of genes inserted into the amyL locus of Bacillus licheniformis has been described in US Patent 5,733,753. To enable plasmid transfer by conjugation, the oriT region of plasmid pUBllO was inserted into pSJ2059 to give plasmid pSJ2773 (fig. 1) . As described in WO 96/23073, mobilization of pUBllO is dependent on a cis acting region (oriT) located 5' to orfβ (Selinger, L. B., McGregor, N. F. , Khachatourians, G. G. , and Hynes, M. F. (1990) . Mobilization of closely related plasmids pUBllO and pBC16 by Bacillus plasmid pXO503 requires trans-acting open reading frame β. J. Bacteriol., 172, 3290-3297). A 550 bp seg- ment from pUBllO, extending from pos. 1020 to pos. 1575 in the pUBllO sequence (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) was PCR amplified using primers LWN5232 and LWN5233.

LWN5232:

5 ' -GTCGGAGCTCATTATTAATCTGTTCAGCAATCGGGC-3 '

LWN5233: 5 ' -GTCGGAGCTCTGCCTTTTAGTCCAGCTGATTTCAC-3 '

The amplified fragment was digested with SacI and initially cloned into the SacI site of an E. coli plasmid (a pUC19 derivative) . The fragment was subsequently excised again using SacI , and cloned into the SacI site of the pUC19 derivative pDN3000 (described in Diderichsen, B., Wedsted, U. , Hedegaard, L. , Jensen, B. R. , Sjøholm, C. (1990). Cloning of aldB , which encodes α-acetolactate decarboxylase, an exoenzyme from Bacillus brevis . J. Bacteriol., 172, 4315-4321), to give pSJ2742 (fig. 2) . The oriT fragment was subsequently excised from pSJ2742 as a 0.56 kb BamHI-Bglll fragment, which was ligated to BamHI digested pSJ2059, and the ligation mixture transformed into competent cells of Bacillus subtilis DN1885 (Diderichsen et al.,

1990) , selecting for resistance to erythromycin (5 μg/ml) and kanamycin (10 μg/ml) at 30 °C. One resulting transformant was SJ2773, containing pSJ2773. B) pSJ4574

The gene encoding "F64L-S65T-GFP", a mutant version of Green Fluorescent Protein was obtained as described in WO 97/11094. The gene was amplified by PCR using primers #109563 and #128360.

#109563:

5 ' -GACTGCATGCGGGGAGGAGAATCATGAGTAAAGGAGAAGAAC-3 '

#128360:

5 ' -CGTGAATTCGAGCTCTGCAGATCCCTTTAGTGTCAATTGG-3 '

The PCR amplified fragment was digested with EcoRI and BspHI . The promoter and ribosome binding site from the Bacillus amy loliquefaciens α-amylase gene, amyQ, was used to enable expression of the "F64L-S65T-GFP" encoding gene. The amyQ promoter was PCR amplified from chromosomal DNA of Bacillus amyloliquefa- ciens using primers LWN8741 and LWN8742.

LWN8741:

5 ' -GTCACTGATCAATCGATTGTTTGAGAAAAGAAG-3 '

LWN8742: 5 ' -GTCACTCATGAGTTTCCTCTCCCTCTC-3 •

The resulting PCR fragment was digested with enzymes BelI and BspHI, and the fragment initially cloned into a pUBllO derived Bacillus vector. The insert was sequenced, and the se- quence found to be identical to the published amyQ promoter sequence (Palva, I., Pettersson, R. F., Kalkkinen, N. , Lehtovaara, P., Sarvas, M. , Sδderlund, H. , Takkinen, K. , Kaariainen, L. (1981) . Nucleotide sequence of the promoter and NH2-terminal signal peptide region of the α-amylase gene from Bacillus amy- loliquefaciens. Gene 15, 43-51) . The promoter was subsequently excised again from this vector, now as a Clal-BspHI fragment.

Cloning vector pUC19 was digested with AccI and EcoRI , and the vector fragment was, in a three-fragment ligation, ligated to the Clal-BspHI fragment containing the amyQ promoter, and the BspHI-_5coRI fragment containing the "F64L-S65T-GFP" encoding gene. Transformants of E. coli SJ2 (Diderichsen et al., 1990) with this ligation mixture were strongly green fluorescent. One such transformant was SJ4574, containing pSJ4574 (fig. 3).

C) pSJ4621

The "F64L-S65T-GFP" encoding gene, expressed from the amyQ promoter, was inserted into the amplification vector plasmid pSJ2773 by the following procedure:

(i) pSJ2773 was digested with Hindlll and AJTIIII, and the 3.95 kb fragment purified.

(ii) pSJ2773 was digested with AJfllll and EcoRI , and the 4.25 kb fragment purified. (iii) pSJ4574 was digested with _5coRI and Hind l , and the 1.0 kb fragment purified.

The three purified fragments were ligated, and the mixture transformed into B. subtilis DN1885, selecting kanamycin (10 μg/ml) and erythromycin (5 μg/ml) resistance at 30°C. Two green flourescent transformants were kept as SJ4621, containing pSJ4621 (fig. 4), and SJ4622, containing pSJ4622.

Construction of con ugative donor strain: The B. subtilis strain PP289-5, described in example 4 of WO 96/23073, was rendered competent and transformed with plasmids pSJ4621 (to give strains SJ4623 and SJ4624) and pSJ4622 (to give strains SJ4625 and SJ4626) . Selection was for erythromycin (5 μg/ml) and tetracycline (5 μg/ml) resistance at 30°C, on plates containing D-alanine (100 μg/ml) .

Plasmid transfer to B. licheniformis :

Strains SJ4623 and SJ4625 were used as donor strains in conjugations, performed essentially as described in WO 96/23073, to transfer the amplification vector plasmid into Bacillus licheniformis recipient strains. These recipient strains contained one chromosomal copy of the alpha-amylase gene, amyL . Transconjugants were obtained that were resistant to erythromycin (5 μg/ml) , to kanamycin (10 μg/ml) , and which expressed F64L-S65T-GFP, as revealed by the greeen fluorescence of the transconjugant colonies.

Chromosomal integration of the amplifiable cassette Transconjugant colonies were streaked on LBPSG plates with erythromycin (5 μg/ml) , and incubated at 50°C, to give rise to colonies in which the amplification vector plasmid had inte- grated into the chromosome. Individual colonies from these 50°C plates were then inoculated into TY medium (10 ml) without antibiotics, and propagated overnight at 30°C, to allow plasmid replication to resume, resulting in the eventual excision of the plasmid from the chromosome and, in the absence of selection, loss of the excised plasmid from the cell. The propagation in TY without antibiotics was repeated once, and cells spread on plates with kanamycin (10 μg/ml) . These were then replica plated onto plates with erythromycin (5 μg/ml) , and kanamycin resistant, erythromycin sensitive colonies isolated. These colonies exhibited, after a few days, a distinct green fluorescence.

Isolation of strains containing several gene copies Bacillus licheniformis strains containing several copies of the genes encoding alpha-amylase, "F64L-S65T-GFP" and kanamycin re- sistance, are isolated from strains constructed as above.

Such strains are isolated by the previously described method of propagation in the presence of increasing concentrations of kanamycin in the growth medium, which selects for survival of strains having multiple gene copies (US 5,733,753). Thus, strains able to grow in the presence of 10 μg/ml, 20 μg/ml, 30 μg/ml, 40 μg/ml, 50 μg/ml, 100 μg/ml and 200 μg/ml are selected.

The yield, in shake flask incubations, of alpha-amylase from the resulting strains is determined. The cellular content of "F64L-S65T-GFP" protein in cells of these cultures is also determined, and a correlation between kanamycin resistance, alpha-amylase yield, and cell fluorescence is established.

In the procedure of the present invention, strains with several gene copies are isolated by a process which does not rely on direct survival selection based on the kanamycin resistance gene, but relies on a physical isolation of cells, which exhibit a higher cellular fluorescence than the population at large. Such a higher cellular fluorescence is obtained if cells contain an increased copy number of the "F64L-S65T-GFP" express- ing gene, consequently this procedure leads to the isolation of cells having multiple gene copies.

If the cell used as a starting point for the isolation of cells with multiple gene copies is one, in which no antibiotic resistance marker gene is present, the resulting cells will con- tain multiple copies of the gene of interest (e.g. an amylase gene) , as well as of the "F64L-S65T-GFP" encoding gene, but will contain no antibiotic resistance genes. See last part of Example 2 herein (vide infra) for further details.

One method to construct such a cell is, in the procedure described above, to utilize an amplification vector plasmid in which the kanamycin resistance gene is flanked by sites recognized by a site-specific recombination enzyme, e.g. the res site of plasmid pAMbetal. Once a cell having the integrated amplification cassette is obtained (indicated by its expression of ka- namycin resistance, of F64L-S65T-GFP, and absence of erythromycin resistance) , the kanamycin resistance gene is removed by introduction into this cell of a vector encoding the pAMbetal re- solvase protein. The technology of utilizing res sites and re- solvase to mediate removal of an integrated antibiotic resis- tance marker gene has been described in details in WO 96/23073. An alternative way of constructing such a cell is described in the following example 2. EXAMPLE 2

Construction of a bacterial strain harbouring an amplifiable cassette comprising a screenable gene encoding green fluorescent protein, but containing no antibiotic resistance marker gene

The strategy in this example is to construct an amplification vector plasmid, which allows the insertion of a promoterless GFP gene immediately downstream of the amyL gene coding sequence of B. licheniformis , followed again by a copy of the amyL promoter region.

Plasmid constructions

A) pSJ4284 and pSJ4285 An about 400 basepair fragment of the amyL gene, encoding the C- terminal part of AmyL, was PCR amplified from chromosomal DNA of Bacillus licheniformis using primers #109561 and #109562.

#109561: 5 ' -GACTGAATTCCAAACATGGTTTAAGCC-3 '

#109562:

5 ' -GACTAAGCTTGGATCCGCATGCCTATCTTTGAACATAAATTG-3 '

The amplified fragment was digested with .EcoRI and Hindlll , and ligated to .EcoRI + HindTII digested plasmid pUC19, to create plasmids pSJ4284 and pSJ4285 (fig. 5) .

B) pSJ4286 and pSJ4287

An about 200 basepair fragment, originating immediately downstream from the amyL gene coding region, was PCR amplified from chromosomal DNA of Bacillus licheniformis using primers #109564 and #109565.

#109564:

5 ' -GACTGAATTCGGATCCGCAGAGAGGACGGATTTCC-3 ' #109565 :

5 ' -GACTAAGCTTCGATACCGTCATTTTCG-3 '

The amplified fragment was digested with .EcoRI and Hin- dill, and ligated to .EcoRI + Hindlll digested plasmid pUC19, to create plasmids pSJ4286 and pSJ4287 (fig. 6) .

C) pSJ4294 and pSJ4295

These plasmids (fig. 7) were created by insertion of the 0.2 kb BamHI-Hindlll fragment, excised from pSJ4286, into BamHI + Hindlll digested pSJ4285.

D) pSJ4313 and pSJ4314

The gene encoding "F64L-S65T-GFP" . The gene was amplified by PCR using primers #109563 and #18842.

#109563: 5 ' -GACTGCATGCGGGGAGGAGAATCATGAGTAAAGGAGAAGAAC-3 '

#18842:

5 ' -CGTGCTCGAGAATTCGGATCCCTTTAGTGTCAATTGG-3 '

The PCR amplified fragment was digested with BamHI and SphI , and inserted into BamHI + SphI digested pSJ4295, to create plasmids pSJ4313 and pSJ4314 (fig. 8) .

E) pSJ4530

To construct the actual amplification vector plasmid, the fol- lowing procedure was used:

(i) pSJ1985 (described in US 5,733,753, example 1 and figure 4) was digested with Bglll and Hindlll, and the 1.7 kb fragment isolated.

(ii) pSJ4313 was digested with BamHI and Hindlll, and the 3.9 kb fragment isolated.

(iii) These two fragments were mixed and ligated, and the liga- tion mixture was subsequently digested with -EcoRI and Hindlll and a fragment of 2.9 kb isolated following agarose gel electro- phoresis.

(iv) This isolated fragment was then ligated to the 5.4 kb .EcoRI-HindiII fragment from pSJ2739 (described in WO 96/23073, 5 example 6) , and the ligation mixture transformed into Bacillus subtilis DN1885 selecting erythromycin (5 μg/ml) at 30°C. Strain SJ4530, thus isolated, contains plasmid pSJ4530 (fig. 9) .

io Construction of coniugative donor strain

The B. subtilis strain PP289-5, described in example 4 of WO 96/23073, was rendered competent and transformed with plasmid pSJ4530, to give strains SJ4541 and SJ4542. Selection was for erythromycin (5 μg/ml) and tetracycline (5 μg/ml) resistance at 15 30°C, on plates containing D-alanine (100 μg/ml) .

Plasmid transfer to B. licheniformis

Strains SJ4541 and SJ4542 are used as donor strains in conjugations, performed essentially as described in WO 96/23073, 20 to transfer the amplification vector plasmid into Bacillus licheniformis recipient strains. These recipient strains contain one chromosomal copy of the alpha-amylase gene, amyL.

Transconjugants are obtained that are resistant to erythromycin (5 μg/ml) .

25

Chromosomal integration of the amplifiable cassette

Transconjugant colonies are streaked on LBPSG plates with erythromycin (5 μg/ml) , and incubated at 50°C, to give rise to colonies in which the amplification vector plasmid has inte-

30 grated into the chromosome. Colonies from these 50°C plates are then inoculated into liquid growth medium without antibiotics, and propagated at 30°C, to allow plasmid replication to resume, resulting in the eventual excision of the plasmid from the chromosome and, in the absence of selection, loss of the excised

35 plasmid from the cell. In the case that the wanted recombination event has taken place, i.e. integration at the AmyL C-terminal coding region and excision at the further downstream region (or vice versa) , such cells contains the F64L-S65T-GFP gene inserted immediately downstream of the amyL gene coding region and can be identified based on their expression of F64L-S65T-GFP protein. Such cells are conveniently isolated from the rest of the popu- lation by means of a flow cytometer with cell sorting capability, such as a FACSCalibur flow cytometer.

Isolation of strains containing several gene copies

Bacillus licheniformis strains containing several copies of the genes encoding alpha-amylase and F64L-S65T-GFP are isolated from strains constructed as above.

A strain constructed as above is the starting culture for propagation in a growth medium, in which F64L-S65T-GFP is expressed. A sample is taken from the culture, and diluted appropriately, typically to between 5 x 10⁵ and 5 x 10⁶ cells/ml.

The diluted sample is analyzed on a FACSCalibur flow cytometer, in which particles are illuminated by the 488 nm light from an argon-ion laser. The FACSCalibur flow cytometer is adjusted so that cells can be detected according to their forward scatter (FSC) , side scatter (SSC) , and green/yellow-green fluorescence at around 530 nm (FL1) signals.

A dot plot is set up with FSC or SSC on one axis, and FL1 on the other axis.

A sort gate corresponding to cells with higher FL1 fluorescence than the cell population at large is set up, and cells with flow cytometric signals within this sort gate are sorted out from the total cell population. The sorted-out cells are plated on LBPSG plates and incubated at 37°C for 1 - 2 days.

Cells are scraped off the plates and pooled. This constitutes a new intermediate cell population.

Using the new intermediate cell population as starting ma- terial, the whole process of propagation, flow cytometry with sorting out of cells with higher FL1 fluorescence than the cell population at large, and incubation of the sorted out-cells to make yet another intermediate cell population, is repeated a number of times, until cell populations with distinctly higher FLl cell fluorescence than the original starting culture can be seen using the FACSCalibur flow cytometer.

From such cell populations with distinctly higher FLl cell fluorescence than the original starting culture, cells are sorted out and plated for single colony isolation on LBPSG plates which are incubated at 37°C for 1-2 days.

Single colonies are compared to the original starting culture by growing the strains in a growth medium that allows ex- pression of both alpha-amylase and F64L-S65T-GFP. When both the FLl cell fluorescence as measured on the FACSCalibur and the alpha-amylase yield is increased compared to the original starting culture, then a strain containing several gene copies have been obtained.

Claims

1. A method of construction a microbial multicopy protein production cell which does not comprise an inserted selection marker gene, comprising a) introducing a sequence into the chromosome of a microbial cell whereby the chromosome of the cell comprises the

DNA structure A-P-S-A, in which

A denotes homologous DNA sequences, P denotes a DNA sequence encoding a protein of interest,

S denotes a DNA sequence encoding a screenable protein; b) propagating said cell; c) screening for a cell which produces increased amounts of said screenable protein; d) recovering the cell identified in c) ; and optionally e) repeating steps b-d using a recovered cell from step d) as starting material.

2. The method according to claim 1, wherein said screenable pro- tein is a protein which is fluorescent under suitable exposure.

3. The method according to claim 2 , wherein said fluorescent protein is Green Fluorescent Protein (GFP) or variants thereof.

4. The method according to claim 2 or 3, wherein the screening in step c) is performed by using a flow cytometer with cell sorting capability, and screening for a cell which has increased content of the fluorescent screenable protein.

5. The method according to claim 4, wherein said fluorescent protein is Green Fluorescent Protein (GFP) or variants thereof, and GFP or variants thereof is made fluorescent under a suitable exposure of light during the screening procedure.

6. The method according to any of claims 1-5, wherein said microbial cell is a bacterial cell.

7. The method according to claim 6, wherein said bacterial cell is a cell of the genus Bacillus, in particular a cell of Bacillus subtilis, Bacillus lentus, Bacillus brevis, Bacillus stearo- thermophilus, Bacillus alkalophilus , Bacillus amyloliquefaciens, Bacillus coagulans , Bacillus circulans , Bacillus lautus, Bacillus thuringiensis , Bacillus clausii, or Bacillus licheniformis.

8. The method of any of the preceding claims, wherein said protein of interest is an enzyme, in particular a protease, a li- pase, an amylase, a galactosidase, a pullanase, a cellulase, a glucose isomerase, a protein disulphide isomerase, a CGT'ase (cyclodextrin gluconotransferase) , a phytase, a glucose oxidase, a glucosyl transferase, a laccase, or a xylanase.

9. A microbial multicopy protein production cell obtainable by any of the methods according to any of claims 1-8 , characterized by that said cell comprises multiple copies of a gene expressing a protein of interest ("P") and multiple copies of a gene expressing a screenable protein ("S") .

10. A process for production of at least one protein of interest in a microbial cell, which method comprises: i) culturing a microbial multicopy protein production cell according to claim 9 under conditions permitting the production of the protein of interest; and ii) recovering said protein of interest from the resulting culture broth or the microbial multicopy protein production cell.