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  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
  • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
  • C12N9/2405—Glucanases
  • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
  • C12N9/2411—Amylases
  • C12N9/2414—Alpha-amylase (3.2.1.1.)
  • C12N9/2417—Alpha-amylase (3.2.1.1.) from microbiological source
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  • C07K14/43595—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from coelenteratae, e.g. medusae
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  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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  • C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
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  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/67—General methods for enhancing the expression
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
  • C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus
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  • C12N15/09—Recombinant DNA-technology
  • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
  • C12N15/90—Stable introduction of foreign DNA into chromosome
  • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination

Definitions

• 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.
• 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)
• the constructed multicopy strains then comprise the structure A-P-M-A-P-M-A-P-M-A .
• 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.
• 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
• 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) .
• GFP Green Fluorescent protein
• 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.
• 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.
• 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
• the final constructed multicopy cell comprises the structure A-P-S-A-P-S-A-P-S-A , and thereby comprises multicopies of the protein of interest "P" .
• 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”) .
• P protein of interest
• S screenable protein
• P multiple copies of a gene expressing a protein of interest
• S 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.
• 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.
• FIGS 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.
• 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 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.
• 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.
• 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”.
• step c) 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 10 5 individual cells are screened, more preferably at least 10 individual cells are screened, and even more preferably are at least 10 7 individual cells are screened.
• 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.
• GFP Green Fluorescent protein
• Green Fluorescent protein 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.
• 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)).
• a flow cytometer with cell sorting capability
• An example of such a flow cytometer is FACSCalibur from Becton Dickinson and Company.
• 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.
• GFP Green Fluorescent protein
• 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.
• Bacillus subtilis Bacillus subtilis
• Bacillus lentus Bacillus lentus
• Bacillus brevis Bacillus stearothermophilus
• Bacillus alkalophilus Bacillus amyloliquefaciens
• Bacillus coagulans Bacillus cir- culans
• Bacillus lautus Bacillus thuringiensis
• the protein of interest 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.
• 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 oxida
• a microbial multicopy protein production cell obtainable by any of the methods according to the invention:
• 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”) .
• 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.
• 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 cells in question.
• culture medium 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
• 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 FACSCaliburTM Systems User's Guide.
• LBPSG agar is LB agar supplemented with phosphate (0.01 M K 3 P0 ) , 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 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.
• the oriT region of plasmid pUBllO was inserted into pSJ2059 to give plasmid pSJ2773 (fig. 1) .
• mobilization of pUBllO is dependent on a cis acting region (oriT) located 5' to orf ⁇ (Selinger, L. B., McGregor, N. F. , Khachatourians, G. G. , and Hynes, M. F. (1990) .
• 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. , Sj ⁇ holm, C. (1990).
• aldB which encodes ⁇ -acetolactate decarboxylase, an exoenzyme from Bacillus brevis . J.
• pSJ2742 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.,
• 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.
• the PCR amplified fragment was digested with EcoRI and BspHI .
• the promoter and ribosome binding site from the Bacillus amy loliquefaciens ⁇ -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.
• 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. , S ⁇ derlund, H. , Takkinen, K. , Kaariainen, L. (1981) .
• 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).
• pSJ2773 was digested with AJfllll and EcoRI , and the 4.25 kb fragment purified.
• 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 ⁇ g/ml) and erythromycin (5 ⁇ g/ml) resistance at 30°C.
• Two green flourescent transformants were kept as SJ4621, containing pSJ4621 (fig. 4), and SJ4622, containing pSJ4622.
• 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 ⁇ g/ml) and tetracycline (5 ⁇ g/ml) resistance at 30°C, on plates containing D-alanine (100 ⁇ g/ml) .
• 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 ⁇ g/ml) , to kanamycin (10 ⁇ g/ml) , and which expressed F64L-S65T-GFP, as revealed by the greeen fluorescence of the transconjugant colonies.
• 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.
• 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).
• strains able to grow in the presence of 10 ⁇ g/ml, 20 ⁇ g/ml, 30 ⁇ g/ml, 40 ⁇ g/ml, 50 ⁇ g/ml, 100 ⁇ g/ml and 200 ⁇ g/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.
• 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.
• 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.
• a site-specific recombination enzyme e.g. the res site of plasmid pAMbetal.
• 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.
• 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.
• 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) .
• 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) .
• the gene encoding "F64L-S65T-GFP” was amplified by PCR using primers #109563 and #18842.
• 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) .
• 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.
• pSJ4313 was digested with BamHI and Hindlll, and the 3.9 kb fragment isolated.
• 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 ⁇ g/ml) and tetracycline (5 ⁇ g/ml) resistance at 15 30°C, on plates containing D-alanine (100 ⁇ g/ml) .
• 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 ⁇ g/ml) .
• Transconjugant colonies are streaked on LBPSG plates with erythromycin (5 ⁇ g/ml) , and incubated at 50°C, to give rise to colonies in which the amplification vector plasmid has inte-
• 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.
• 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 10 5 and 5 x 10 6 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.
• FSC forward scatter
• SSC side scatter
• FL1 green/yellow-green fluorescence at around 530 nm
• 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.
• 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.
• 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.
• a strain containing several gene copies have been obtained.

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