Patent Publication Number: US-2023151347-A1

Title: A genetically modified bacillus subtilis strain, optimized vectors, and uses thereof

Description:
The present invention relates to a genetically modified  Bacillus subtilis  strain which has been transformed with an optimized vector, mainly for producing a D-psicose 3-epimerase. 
     D-psicose, also called D-allulose, is a rare sugar epimer of fructose. It can be found in nature but at very low concentrations like in edible mushrooms, in jackfruit, in wheat and in  Itea  plants. 
     At the opposite of fructose, the metabolism of psicose in humans is partly absorbed and metabolized in energy, and partly excreted unchanged in the urine and in the faeces. 
     D-psicose has a noncaloric nature, a sweet taste equivalent to sucrose, a positive effect on the reduction of the glycemic response, an antiobesity effect, and the like. It is then particularly useful for preventing lifestyle-related diseases, such as diabetes or obesity. 
     D-psicose is very difficult to chemically synthetize. Therefore, interconversion between D-fructose and D-psicose by epimerization using the enzymes named D-psicose 3-epimerases has been considered as an attractive way of D-psicose production. 
     In that purpose, it has been provided improved variants of D-psicose 3-epimerase which are weak-acid stable, thermostable, and which have higher catalysis efficiency and turnover for the substrate D-fructose (PCT/EP2014/068628). This international application also discloses a host cell (such as  Escherichia coli  or  Bacillus subtilis ) having a nucleic acid coding for the said improved variants of D-psicose 3-epimerase. 
     Another strategy has been to clone and express the D-psicose-3-epimerase from  Clostridium cellulolyticum  in  Escherichia coli  (Cloning, Expression, and Characterization of a D-psicose-3-epimerase from  Clostridium cellulolyticum  H10, Journal of Agricultural and Food Chemistry, 2011, 59, 7785-7792, Wanmeng Fu et al.). 
     It has also been disclosed the cloning and expression of D-psicose-3-epimerase from  Clostridium scindens  (ATCC 35704) in  Bacillus subtilis . The selection of the recombinant strains of  Bacillus subtilis  which have been transformed with a plasmid expressing the gene coding for D-psicose-3-epimerase is based on D-alanine defective selection marker (CN104894047). 
     It is appeared however to the inventors of the present invention that these strategies were not appropriate for industrial application, notably because of the low activity of the enzyme expression systems in the strains of  Bacillus subtilis.    
     Therefore, there is still a need for improved D-psicose-3-epimerase production, as well as a need for improved D-psicose production. The methods have to be appropriate for industrial application and cost-effective. The methods have also to comply with safety and environment regulations. 
     Thus, the present invention aims to provide a method for improving D-psicose-3-epimerase production, as well as a method for improving D-psicose production, which are appropriate for industrial application, cost-effective, and which comply with safety and environment regulations. 
     The present invention relies on the unexpected results of the inventors showing that for improving D-psicose-3-epimerase production, as well as D-psicose production, it was necessary (i) not only to develop an optimized strain of  Bacillus subtilis , but also (ii) to develop an optimized vector for higher D-psicose-3-epimerase expression. 
     The present invention also relies on the unexpected results of the inventors relative to an optimized fermentation medium for higher D-psicose-3-epimerase expression. 
     The objects of the present invention are therefore an optimized  Bacillus subtilis  strain, an optimized nucleic acid molecule comprising a nucleic acid sequence coding for D-psicose 3-epimerase, an optimized recombinant expression vector, an optimized recombinant host cell, and uses thereof in a method for producing a D-psicose 3-epimerase and in a method for producing D-psicose. The methods of obtaining the optimized and recombinant  Bacillus subtilis  strains are also an object of the present invention, as well as the optimized fermentation medium. 
     In a first aspect, the present invention relates thus to a genetically modified  Bacillus subtilis  strain wherein the alanine racemase alrA gene is inactivated, and having at least a further gene inactivation chosen among the inactivation of the sporulation yfqD gene, and/or the inactivation of the erythromycin resistance EmR-comK gene cassette. 
     The term “ Bacillus subtilis  strain” according to the invention means any strains of bacteria belonging to the genus  Bacillus  and the species  subtilis . Cells of these organisms are less than 1 μm wide, sporangia are not swollen, and spores are ellipsoidal.  Bacillus subtilis  can be identified by several methods, such as the one described in Biochemical Test and Identification of  Bacillus subtilis , Aryal S. 2016. http://www.microbiologyinfo.com/biochemical-test-andidentifiication-of- bacillus - subtilis /. In an embodiment of the invention, the “ Bacillus subtilis  strain” is isolated and/or purified. 
     The term “alanine racemase alrA gene” according to the invention means the gene coding for the enzyme D-alanine racemase, such enzyme catalyzing the chemical reaction from L-alanine to D-alanine. The “alrA” gene is also named “dal” gene, and is represented by SEQ ID NO: 17. SEQ ID NO: 17 (1.17 kb DNA fragment) contains the entire alrA structural gene (coding the D-alanine racemase identified in GenBank, under the number CAB12271.1) and regulatory signals for its expression. Within a large part of the bacteria, D-alanine is an important component of the glycan subunits to form the cell wall (composed of peptidoglycans). Alanine is usually found as the L-stereoisomer in nature, making the conversion to D-alanine by the cytoplasmic D-alanine racemase (alrA) essential for cell growth. Lack of the enzyme leads to rapid cell lysis due to a failure in the initial step of peptidoglycan biosynthesis. According to the invention, the genetically modified  Bacillus subtilis  strain is intended to be transformed with a vector in which the D-alanine racemase gene has been inserted. Therefore a  Bacillus subtilis  strain, in which the alrA gene is deleted (meaning that the  Bacillus subtilis  is “D-alanine defective”), and which has been successfully transformed with the said vector is able to grow without D-alanine supplementation. The main advantage of this strategy is to provide direct selection for the recombinant  Bacillus subtilis  in complex media without antibiotics. Moreover, as the D-alanine racemase is involved in the cell wall metabolism, the loss of the activity leads to the cell lysis, preventing the accumulation of a population of  Bacillus subtilis  (cells) which have lost the vector. In the present invention, the terms “alrA gene”, “dal gene”, “alanine racemase gene”, alanine racemase alrA gene and “D-alanine racemase gene” can be used instead of another. 
     The term “sporulation yfqD gene” according to the invention means the gene which acts during the stage IV of the endospore maturation. The exact function of this gene is unknown, but its inactivation/deletion leads to a complete sporulation abortion. This “yfqD gene” is represented by SEQ ID NO: 18.  Bacillus  genus bacteria are known to produce a dedicated, very resistant and non-reproductive structure to enter in a state of dormancy: the endospores. Bacterial endospores keeps all material the cell needs to recover a living cell when favorable conditions will appear. The endospores are the perfect dissemination factor for the strain and their formation is a serious risk for environmental and health contamination. It is important to have a strain wherein the endospore forming pathway is aborted, notably for  Bacillus  strain which are intended to be used for industrial application. Therefore, a  Bacillus subtilis  strain wherein the sporulation yfqD gene is deleted complies with safety and environment regulations. To determine if a strain is sporulation deficient, a heat treatment can be applied to the strain; if the strain can produce bright spores then the strain is not sporulation deficient, whereas if the strain cannot produce bright spores then the strain is sporulation deficient. 
     The term “erythromycin resistance EmR-comK gene cassette” means a cassette containing the EmR gene and the comK gene. Surprisingly, it has indeed been found by the inventors that some  Bacillus subtilis  strain are resistant to erythromycin. In the  Bacillus subtilis  strain of the present invention, the EmR-comK gene cassette is inactivated, notably removed. Then, the “deletion of erythromycin resistance EmR-comK gene cassette” means the “removal of erythromycin resistance EmR-comK gene cassette”. The above-mentioned cassette is represented by SEQ ID NO: 19. To determine if a strain is resistant or sensitive to erythromycin, the following test can be applied: contacting the strain with high concentration of erythromycin (for example 5 μg/mL); if the strain is still able to cultivate then the strain is resistant to erythromycin, whereas if the strain is not able to cultivate then the strain is sensitive to erythromycin. Therefore, a  Bacillus subtilis  strain wherein the erythromycin resistance gene is deleted complies with safety and environment regulations. 
     In an embodiment, the present invention relates thus to a genetically modified  Bacillus subtilis  strain wherein the alanine racemase alrA gene represented by SEQ ID NO: 17 or a sequence having at least 80% of identity with SEQ ID NO: 17 is inactivated, and having at least a further gene inactivation chosen among the inactivation of the sporulation yfqD gene represented by SEQ ID NO: 18 or a sequence having at least 80% of identity with SEQ ID NO: 18, and/or the inactivation of the erythromycin resistance EmR-comK gene cassette represented by SEQ ID NO: 19 or a sequence having at least 80% of identity with SEQ ID NO: 19. The percentage of identity between two sequences (A) and (B) can be obtained by dividing the full number of identical amino acid residues aligned by the full number of residues contained in the longest sequence between the sequence (A) and (B). Said alignment of sequences can be carried out by well-known methods, for example using the algorithm for global alignment of Needleman Wunsch. The term “at least 80% of identity” means 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 and 100% of identity, notably 90%, preferably 95% and even more preferably 99% with SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19. 
     The term “inactivated” and “gene inactivation” according to the invention means that the gene is deleted or inactivated by one or several mutations. The mutagenesis may be site-directed and/or random. The mutagenesis can be insertion, deletion, substitution of one or several nucleotides. In a preferred embodiment, “inactivated” and “gene inactivation” means that the gene is deleted. In another preferred embodiment, it means that the locus is deleted. In a preferred embodiment, the gene(s) is/are knocked-out. Deletion of the gene can be achieved by any technics known from the skilled person, for example a gene can be knocked-out by the Cre-Lox system, by any other site-specific recombinase systems (for example FLP, Dre) or by analogous methods such as MazF based system (i.e. by using a MazF cassette). 
     In an embodiment, the genetically modified  Bacillus subtilis  strain is a strain wherein the alanine racemase alrA gene and the sporulation yfqD gene are inactivated, notably by a deletion of the genes. An example of such a  Bacillus subtilis  strain is the strain which has been deposited at the National Collection of Microorganisms Cultures on Oct. 18, 2017 under the accession number CNCM I-5252. This strain is called BsR4 in the example of the present invention. 
     In another embodiment, the genetically modified  Bacillus subtilis  strain is a strain wherein the alanine racemase alrA gene and the erythromycin resistance EmR-comK gene cassette are inactivated, notably by a deletion of the genes. An example of such a  Bacillus subtilis  strain is the strain which has been deposited at the National Collection of Microorganisms Cultures on Oct. 18, 2017 under the accession number CNCM I-5251. This strain is called BsR3 in the example of the present invention. 
     In another and preferred embodiment, the genetically modified  Bacillus subtilis  strain is a strain wherein the alanine racemase alrA gene, the erythromycin resistance EmR-comK gene cassette, and the sporulation yfqD gene are inactivated, notably by a deletion of the genes. An example of such a strain is the strain which has been deposited at the National Collection of Microorganisms Cultures on Oct. 18, 2017 under the accession number CNCM I-5253. This strain is called BsR5 in the example of the present invention. 
     The above-mentioned strains BsR3, BsR4 and BsR5 have been deposited at the National Collection of Microorganisms Cultures of the Pasteur Institute, located at Institut Pasteur, 25, 28 rue du Docteur Roux, 75724 Paris Cedex 15, France. 
     In a second aspect, the present invention relates to a method of obtaining a genetically modified  Bacillus subtilis  strain as mentioned above, comprising mutagenesis or genetic transformation of a  Bacillus subtilis  strain. Notably, such method allows obtaining the strains BsR3, BsR4 and BsR5. 
     The term “genetic transformation” according to the present invention means notably genes deletion. 
     In an embodiment, the present invention relates thus to a method of obtaining a  Bacillus subtilis  which is D-alanine defective (alrA − ) and erythromycin sensitive and/or sporulation deficient, preferably a  Bacillus subtilis  which is D-alanine defective (alrA − ) and erythromycin sensitive and sporulation deficient. 
     In an embodiment, the said method of obtaining a genetically modified  Bacillus subtilis  strain, notably the strain BsR4, comprises the following steps:
         (a) the alanine racemase alrA gene is deleted in a  Bacillus subtilis , preferably by a Cre/Lox system, in order to provide a D-alanine defective  Bacillus subtilis  (alrA−);   (b) the sporulation yfqD gene is deleted, preferably by using a MazF based system, in order to provide a  Bacillus subtilis  which is sporulation deficient, and D-alanine defective (alrA − ).       

     In this embodiment, the step (b) is preferably performed on  Bacillus subtilis  strain obtained in step (a). In an embodiment, the strain obtained in step (b) is erythromycin sensitive or erythromycin resistant, preferably erythromycin resistant. 
     A  Bacillus subtilis  which is sporulation deficient, and D-alanine defective (alrA − ) can be obtained, for example, as described in Example 3.2.a. 
     In another embodiment, the said method of obtaining a genetically modified  Bacillus subtilis  strain, notably the strain BsR3, comprises the following steps:
         (a) the alanine racemase alrA gene is deleted in a  Bacillus subtilis , preferably by a Cre/Lox system, in order to provide a D-alanine defective  Bacillus subtilis  (alrA−);   (b) the erythromycin resistance EmR-comK gene cassette is removed/deleted, preferably by using a MazF based system, in order to provide an erythromycin sensitive and a D-alanine defective  Bacillus subtilis  (alrA − ).       

     In this embodiment, the step (b) is preferably performed on  Bacillus subtilis  strain obtained in step (a). In an embodiment, the strain obtained in step (b) is sporulation deficient or sporulation efficient, preferably sporulation deficient. 
     A  Bacillus subtilis  which is erythromycin sensitive, and D-alanine defective (alrA − ) can be obtained, for example, as described in Example 3.1. 
     In a preferred and another embodiment, the said method of obtaining a genetically modified  Bacillus subtilis  strain, notably the strain BsR5, comprises the following steps:
         (a) the alanine racemase alrA gene is deleted in a  Bacillus subtilis , preferably by a Cre/Lox system, in order to provide a D-alanine defective  Bacillus subtilis  (alrA−);   (b) the erythromycin resistance EmR-comK gene cassette is removed/deleted, preferably by using a MazF based system, in order to provide an erythromycin sensitive and a D-alanine defective  Bacillus subtilis  (alrA − );   (c) the sporulation yfqD gene is deleted, preferably by using a MazF based system, in order to provide a  Bacillus subtilis  which is erythromycin sensitive, sporulation deficient, and D-alanine defective (alrA − ).       

     A  Bacillus subtilis  which is erythromycin sensitive, sporulation deficient, and D-alanine defective (alrA − ) can be obtained, for example, as described in Example 3.2.b. 
     In this embodiment, the step (b) is preferably performed on  Bacillus subtilis  strain obtained in step (a) and the step (c) is preferably performed on  Bacillus subtilis  strain obtained in step (b). In another embodiment, the deletion of the sporulation yfqD gene can be performed before the deletion of the erythromycin resistance EmR-comK gene cassette. 
     In a third aspect, the present invention relates to an isolated nucleic acid molecule comprising a nucleic acid sequence coding for D-psicose 3-epimerase and a sequence comprising or consisting of SEQ ID NO: 1 or of SEQ ID NO: 2. 
     SEQ ID NO: 1 and SEQ ID NO: 2 correspond to sequence of optimized 5′ untranslated region (5′ UTR) for D-psicose 3-epimerase expression. Such sequences are upstream of the nucleic acid sequence coding for D-psicose 3-epimerase. In a preferred embodiment, SEQ ID NO: 1 or SEQ ID NO: 2 are directly upstream of the ATG codon of nucleic acid sequence coding for D-psicose 3-epimerase. In that embodiment, the last base of SEQ ID NO: 1 or SEQ ID NO: 2 is then followed by the first base of the ATG codon of nucleic acid sequence coding for D-psicose 3-epimerase. Sequences comprising or consisting of SEQ ID NO: 1 or of SEQ ID NO: 2 are operably linked to the nucleic acid sequence coding for D-psicose 3-epimerase. The term “operably linked” according to the invention means that sequences comprising or consisting of SEQ ID NO: 1 or of SEQ ID NO: 2 is attached or linked to the sequence coding for D-psicose 3-epimerase in such a manner as to allow these sequences comprising or consisting of SEQ ID NO: 1 or of SEQ ID NO: 2 to control the expression of D-psicose 3-epimerase. SEQ ID NO: 1 or SEQ ID NO: 2 are non-coding sequences, contrary to nucleic acid sequence coding for D-psicose 3-epimerase. More precisely, SEQ ID NO: 1 or SEQ ID NO: 2 are optimized ribosome binding sites. 
     The term “D-psicose 3-epimerase” or “DPEase” according to the invention refers to the ketose 3-epimerase whose D-psicose is the optimum substrate. It refers to an enzyme which has the ability to modify D-fructose into D-psicose. 
     In a preferred embodiment, the present invention relates to an isolated nucleic acid molecule comprising a nucleic acid sequence coding for D-psicose 3-epimerase and a sequence comprising or consisting of SEQ ID NO: 2. 
     In an embodiment, the nucleic acid sequence coding for D-psicose 3-epimerase is chosen among the nucleic acid of SEQ ID NO: 3, SEQ ID NO:4 or the nucleic acid coding for SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13, and is preferably SEQ ID NO: 4. SEQ ID NO: 5 to SEQ ID NO: 13 correspond to the nucleic acid coding for the optimized variants disclosed in PCT/EP2014/068628, i.e optimized variants having a serine residue at position 211. 
     The term “nucleic acid” according to the invention may be DNA or RNA. The term “DNA” includes cDNA, gDNA or artificially synthetized DNA. The DNA may be single strand or double strand. In a preferred embodiment, the nucleic acid of the present invention is DNA. It will be understood that as a result of the degeneracy of the genetic code, a multitude of nucleotide sequences may code a given protein. In an embodiment, the said nucleic acid molecule is artificial. 
     According to the present invention, the nucleic acid coding for D-psicose 3-epimerase can be present in the host cell as an episomic sequence or can be incorporated into its chromosome. The nucleic acid coding for D-psicose 3-epimerase can also be present in the host cell in one copy or in several copies. 
     The present invention also relates to an expression cassette of a nucleic acid molecule as mentioned above. In that embodiment, this expression cassette comprises all elements required for expression of D-psicose 3-epimerase, in particular all the elements required for transcription and translation in the host cell. 
     In a fourth aspect, the present invention relates to a recombinant expression vector comprising a nucleic acid molecule as mentioned above, or an expression cassette of a nucleic acid molecule as mentioned above. In another embodiment, the said recombinant expression vector comprises or consists of SEQ ID NO: 14, SEQ ID NO: 15 or SEQ ID NO: 16. 
     The term “a recombinant expression vector” means a vector which comprises the elements required/necessary for its expression, namely which allows expressing the D-psicose 3-epimerase in the host cell. Preferably the vector is a self-replicable vector. In particular, the vector or the expression cassette also comprises a promoter sequence (for example the promotor P43), a terminator sequence and optionally an enhancer. 
     A “vector” according to the invention can be a plasmid, a phage, a phagemid, a cosmid, a virus, YAC, BAC, . . . . In a preferred embodiment the vector is a plasmid. In a preferred embodiment, the vector is an integration vector suitable to incorporate the sequence coding for D-psicose 3-epimerase into the chromosome of the host cell. More preferably, the recombinant expression vector of the invention comprises or consists of SEQ ID NO: 16. 
     In a fifth aspect, the present invention relates to a recombinant host cell comprising a nucleic acid as above-mentioned, or a recombinant expression vector as above-mentioned. 
     The term “host cell” according to the invention can be a prokaryote or a eukaryote host cell. In a particular embodiment, the host cell is a GRAS (Generally Recognized As Safe) strain, more preferably  Bacillus subtilis  strain. In a preferred embodiment, the host cell is a genetically modified  Bacillus subtilis  strain as defined above. 
     In an embodiment, the cell is non-human and non-embryonic. 
     In an embodiment, the host cell is cultured under conditions such that the D-psicose 3-epimerase is expressed by the host cell. In a preferred embodiment, the D-psicose 3-epimerase is recovered from the culture media. 
     In a preferred embodiment, the present invention relates to a recombinant host cell comprising a recombinant expression vector comprising or consisting of SEQ ID NO: 16. 
     In an embodiment, the host cell is a genetically modified  Bacillus subtilis  strain deposited at the National Collection of Microorganisms Cultures on Oct. 18, 2017 under the Number CNCM I-5251 which comprises a nucleic acid comprising or consisting of SEQ ID NO: 14. This refers to the strain called BsR3 which has been transformed with the plasmid called pR1. 
     In another embodiment, the host cell is a genetically modified  Bacillus subtilis  strain deposited at the National Collection of Microorganisms Cultures on Oct. 18, 2017 under the Number CNCM I-5251 which comprises a nucleic acid comprising or consisting of SEQ ID NO: 15. This refers to the strain called BsR3 which has been transformed with the plasmid called pR2. 
     In another embodiment, the host cell is a genetically modified  Bacillus subtilis  strain deposited at the National Collection of Microorganisms Cultures on Oct. 18, 2017 under the Number CNCM I-5251 which comprises a nucleic acid comprising or consisting of SEQ ID NO: 16. This refers to the strain called BsR3 which has been transformed with the plasmid called pR3. 
     In another embodiment, the host cell is a genetically modified  Bacillus subtilis  strain deposited at the National Collection of Microorganisms Cultures on Oct. 18, 2017 under the Number CNCM I-5252 which comprises a nucleic acid comprising or consisting of SEQ ID NO: 14. This refers to the strain called BsR4 which has been transformed with the plasmid called pR1. 
     In another embodiment, the host cell is a genetically modified  Bacillus subtilis  strain deposited at the National Collection of Microorganisms Cultures on Oct. 18, 2017 under the Number CNCM I-5252 which comprises a nucleic acid comprising or consisting of SEQ ID NO: 15. This refers to the strain called BsR4 which has been transformed with the plasmid called pR2. 
     In another embodiment, the host cell is a genetically modified  Bacillus subtilis  strain deposited at the National Collection of Microorganisms Cultures on Oct. 18, 2017 under the Number CNCM I-5252 which comprises a nucleic acid comprising or consisting of SEQ ID NO: 16. This refers to the strain called BsR4 which has been transformed with the plasmid called pR3. 
     In another embodiment, the host cell is a genetically modified  Bacillus subtilis  strain deposited at the National Collection of Microorganisms Cultures on Oct. 18, 2017 under the Number CNCM I-5253 which comprises a nucleic acid comprising or consisting of SEQ ID NO: 14. This refers to the strain called BsR5 which has been transformed with the plasmid called pR1. 
     In another embodiment, the host cell is a genetically modified  Bacillus subtilis  strain deposited at the National Collection of Microorganisms Cultures on Oct. 18, 2017 under the Number CNCM I-5253 which comprises a nucleic acid comprising or consisting of SEQ ID NO: 15. This refers to the strain called BsR5 which has been transformed with the plasmid called pR2. 
     In another and preferred embodiment, the host cell is a genetically modified  Bacillus subtilis  strain deposited at the National Collection of Microorganisms Cultures on Oct. 18, 2017 under the Number CNCM I-5253 which comprises a nucleic acid comprising or consisting of SEQ ID NO: 16. This refers to the strain called BsR5 which has been transformed with the plasmid called pR3. 
     The term “a host cell which is a genetically modified  Bacillus subtilis  strain and which comprises a nucleic acid” means that the said genetically modified  Bacillus subtilis  strain has been transformed with a nucleic acid or with a vector comprising a nucleic acid. As used herein, the terms “transformed” can means “stably transformed” and refers to a cell into which a nucleotide sequence has been introduced by human intervention. The term “transform” or “transforming” or “transformed” can also be understood by meaning “modification” or “modifying” or “modified”; but also meaning “transfection” or “transfecting” or “transfected” and “transduction” or “transducing” or “transduced” according to the used vector. 
     In a sixth aspect, the present invention relates to a method of obtaining a recombinant  Bacillus subtilis  expressing D-psicose 3-epimerase, as mentioned above, comprises the following steps:
         (a) obtaining a genetically modified  Bacillus subtilis  strain wherein the alanine racemase alrA gene is inactivated, and having at least a further gene inactivation chosen among the inactivation of the sporulation yfqD gene, and/or the inactivation of the erythromycin resistance EmR-comK gene cassette;   (b) transforming the said genetically modified  Bacillus subtilis  obtained in step (a) with a vector comprising a nucleic acid molecule comprising a nucleic acid sequence coding for D-psicose 3-epimerase and a sequence comprising or consisting of SEQ ID NO: 1 or of SEQ ID NO: 2.       

     In an embodiment, the method of obtaining a recombinant  Bacillus subtilis  expressing D-psicose 3-epimerase, as mentioned above, comprises the following steps:
         (a) obtaining a genetically modified  Bacillus subtilis  strain wherein the alanine racemase alrA gene and the sporulation yfqD gene are inactivated;   (b) transforming the said genetically modified  Bacillus subtilis  obtained in step (a) with a vector comprising a nucleic acid molecule comprising a nucleic acid sequence coding for D-psicose 3-epimerase and a sequence comprising or consisting of SEQ ID NO: 1 or of SEQ ID NO: 2.       

     In an embodiment, the method of obtaining a recombinant  Bacillus subtilis  expressing D-psicose 3-epimerase, as mentioned above, comprises the following steps:
         (a) obtaining a genetically modified  Bacillus subtilis  strain wherein the alanine racemase alrA gene and the erythromycin resistance EmR-comK gene cassette are inactivated;   (b) transforming the said genetically modified  Bacillus subtilis  obtained in step (a) with a vector comprising a nucleic acid molecule comprising a nucleic acid sequence coding for D-psicose 3-epimerase and a sequence comprising or consisting of SEQ ID NO: 1 or of SEQ ID NO: 2.       

     In a preferred embodiment, the method of obtaining a recombinant  Bacillus subtilis  expressing D-psicose 3-epimerase, as mentioned above, comprises the following steps:
         (a) obtaining a genetically modified  Bacillus subtilis  strain wherein the alanine racemase alrA gene, the erythromycin resistance EmR-comK gene cassette, and the sporulation yfqD gene are inactivated;   (b) transforming the said genetically modified  Bacillus subtilis  obtained in step (a) with a vector comprising a nucleic acid molecule comprising a nucleic acid sequence coding for D-psicose 3-epimerase and a sequence comprising or consisting of SEQ ID NO: 1 or of SEQ ID NO: 2.       

     In a preferred embodiment, the method of obtaining a recombinant  Bacillus subtilis  expressing D-psicose 3-epimerase, as mentioned above, comprises the following steps:
         (a) obtaining a genetically modified  Bacillus subtilis  strain wherein the alanine racemase alrA gene, the erythromycin resistance EmR-comK gene cassette, and the sporulation yfqD gene are inactivated;   (b) transforming the said genetically modified  Bacillus subtilis  obtained in step (a) with a vector comprising or consisting of SEQ ID NO: 16.       

     In a preferred embodiment, the method of obtaining a recombinant  Bacillus subtilis  expressing D-psicose 3-epimerase, as mentioned above, comprises the following steps:
         (a) deleting the alanine racemase alrA gene in a  Bacillus subtilis , preferably by a Cre/Lox system, in order to provide a D-alanine defective  Bacillus subtilis  (alrA − );   (b) deleting the erythromycin resistance EmR-comK gene cassette in the  Bacillus subtilis  strain obtained in step (a), preferably by using a MazF based system, in order to provide an erythromycin sensitive and a D-alanine defective  Bacillus subtilis  (alrA − );   (c) deleting the sporulation yfqD gene in the  Bacillus subtilis  strain obtained in step (b), preferably by using a MazF based system, in order to provide a  Bacillus subtilis  which is erythromycin sensitive, sporulation deficient, and D-alanine defective (alrA − );   (d) transforming the said genetically modified  Bacillus subtilis  obtained in step (c) with a vector comprising or consisting of SEQ ID NO: 16.       

     In a seventh aspect, the present invention relates to a method for producing a D-psicose 3-epimerase, notably by a fermentation process, comprising culturing the recombinant host cell as mentioned above, and optionally recovering the produced D-psicose 3-epimerase from the resulting culture. 
     The present invention also relates to the use of a nucleic acid, an expression cassette, an expression vector, or a host cell as mentioned above for producing a D-psicose 3-epimerase according to the present invention. 
     In an embodiment, such method for producing a D-psicose 3-epimerase comprises the following steps:
         culturing the recombinant host cell as mentioned above in a suitable culture medium comprising a sugar concentration of at least 60 g/L, notably 60 g/L;   and optionally recovering the produced D-psicose 3-epimerase from the resulting culture.       

     In an embodiment, the suitable culture medium is a suitable fermentation medium. 
     In a preferred embodiment, the sugar is the glucose. The inventors of the present invention have also surprisingly found that the use of a glucose concentration of about 60 g/L is an optimized concentration for the production of D-psicose 3-epimerase according to the present invention. This quantity is particularly adapted for a batch of 20 L, and will be adapted if necessary for other batches. Other components of suitable medium will be apparent to skilled person. For example an appropriate medium can also comprises yeast, KH 2 PO 4 , MgSO 4 , 2H 2 O, MnSO 4 , H 2 O, . . . . Advantageously, a culture medium contains a carbon source (such as glucose), a nitrogen source (such as yeast, yeast extract(s) or amino acids), salts (such as ammonium sulfate, micronutrients (such as iron and magnesium salt), and organic vitamins if necessary. Other specific culture conditions, such as temperature, pH and the like, may be those that are used for the host cell selected for expression, and will be apparent to skilled person. For example, the temperature may be above 30° C. (notably 36.5-37.5° C.) and pH around 6. 
     In a preferred embodiment, culturing is carried out in batch culture. 
     In a preferred embodiment, the host cell used in the method for producing a D-psicose 3-epimerase is the genetically modified  Bacillus subtilis  strain deposited at the National Collection of Microorganisms Cultures on Oct. 18, 2017 under the Number CNCM I-5253 which comprises a nucleic acid comprising or consisting of SEQ ID NO: 16 (i.e. the strain called BsR5 which has been transformed with the plasmid called pR3). 
     In an eighth aspect, the present invention relates to the use of a D-psicose 3-epimerase obtained according to the present invention for producing D-psicose. 
     In an embodiment, the present invention relates to a method for producing a D-psicose comprising:
         (a) culturing the recombinant host cell as defined above;   (b) recovering the produced D-psicose 3-epimerase from the resulting culture;   (c) contacting the D-psicose 3-epimerase obtained in step (b) with D-fructose in conditions suitable for D-psicose 3-epimerase activity; and   (d) optionally recovering the produced D-psicose.       

     In a preferred embodiment, the recombinant host cell used in the method for producing a D-psicose is the genetically modified  Bacillus subtilis  strain deposited at the National Collection of Microorganisms Cultures on Oct. 18, 2017 under the Number CNCM I-5253 which comprises a nucleic acid comprising or consisting of SEQ ID NO: 16 (i.e. the strain called BsR5 which has been transformed with the plasmid called pR3). 
     Suitable conditions for producing D-psicose can be defined by the skilled person. 
     The Table 1 below mentions the sequences used in the present invention. 
     
       
         
           
               
               
               
             
               
                   
                   
               
               
                   
                 Sequence number 
                 Sequences 
               
               
                   
                   
               
             
            
               
                   
                 SEQ ID NO: 1, 
                 AGAAAGGAGGATTACAT 
               
               
                   
                 optimized ribosome 
                   
               
               
                   
                 binding sites 
                   
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 2, 
                 AGAAAGGAGGATTCGAA 
               
               
                   
                 optimized 
                   
               
               
                   
                 translation 
                   
               
               
                   
                 initiation region 
                   
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 3, 
                 ATGAAACATGGTATATACTACGCATATTGGGAACAAGAATGGGAAGCTGATTACAAATACTATATTGAGAAGGTTGCA 
               
               
                   
                 nucleic acid 
                 AAGCTTGGTTTTGATATTCTAGAGATTGCAGCTTCACCGCTACCTTTTTACAGTGACATTCAGATTAATGAGCTCAAG 
               
               
                   
                 coding for DPEase 
                 GCATGTGCCCATGGCAATGGAATTACACTTACGGTAGGCCATGGGCCTAGTGCAGAACAAAACCTGTCTTCTCCCGAC 
               
               
                   
                 H10 from 
                 CCCGATATTCGCAAAAATGCTAAAGCTTTTTATACCGATTTACTCAAACGACTTTACAAGCTGGATGTACATTTGATA 
               
               
                   
                 literature 
                 GGTGGGGCTTTATATTCTTATTGGCCGATAGATTACACAAAGACAATTGATAAAAAAGGCGATTGGGAACGCAGCGTT 
               
               
                   
                   
                 GAAAGTGTTCGAGAAGTTGCTAAGGTGGCCGAAGCCTGTGGAGTGGATTTCTGCCTAGAGGTTCTTAATAGATTTGAG 
               
               
                   
                   
                 AATTATTTAATTAACACAGCACAAGAGGGTGTAGATTTTGTAAAACAGGTTGACCATAACAATGTAAAGGTAATGCTT 
               
               
                   
                   
                 GATACCTTCCATATGAATATTGAGGAAGATAGTATCGGAGGTGCAATCAGGACTGCGGGCTCTTACTTGGGACATTTA 
               
               
                   
                   
                 CACACTGGCGAATGTAATCGTAAAGTTCCCGGCAGAGGAAGAATTCCATGGGTAGAAATTGGTGAGGCTCTTGCTGAC 
               
               
                   
                   
                 ATAGGTTATAACGGTAGTGTTGTTATGGAACCTTTTGTTAGAATGGGCGGAACTGTCGGATCTAATATTAAGGTTTGG 
               
               
                   
                   
                 CGTGACATTAGTAACGGTGCAGATGAGAAAATGCTGGATAGAGAAGCACAGGCCGCACTTGATTTCTCCAGATATGTA 
               
               
                   
                   
                 TTAGAATGTCATAAACACTCCTGA 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 4, 
                   CATATG AAACATGGTATATACTACGCATATTGGGAACAAGAATGGGAAGCTGATTACAAATACTATATTGAGAAGGTT 
               
               
                   
                 nucleic acid 
                 GCAAAGCTTGGTTTTGATATTCTAGAGATTGCAGCTTCACCGCTACCTTTTTACAGTGACATTCAGATTAATGAGCTC 
               
               
                   
                 coding for 
                 AAGGCATGTGCCCATGGCAATGGAATTACACTTACGGTAGGCCATGGGCCTAGTGCAGAACAAAACCTGTCTTCTCCC 
               
               
                   
                 DPEase H10 de novo 
                 GACCCCGATATTCGCAAAAATGCTAAAGCTTTTTATACCGATTTACTCAAACGACTTTACAAGCTGGATGTACATTTG 
               
               
                   
                 synthetized 
                 ATAGGTGGGGCTTTATATTCTTATTGGCCGATAGATTACACAAAGACAATTGATAAAAAAGGCGATTGGGAACGCAGC 
               
               
                   
                   
                 GTTGAAAGTGTTCGAGAAGTTGCTAAGGTGGCCGAAGCCTGTGGAGTGGATTTCTGCCTAGAGGTTCTTAATAGATTT 
               
               
                   
                   
                 GAGAATTATTTAATTAACACAGCACAAGAGGGTGTAGATTTTGTAAAACAGGTTGACCATAACAATGTAAAGGTAATG 
               
               
                   
                   
                 CTTGATACCTTCCACATGAATATTGAGGAAGATAGTATCGGAGGTGCAATCAGGACTGCGGGCTCTTACTTGGGACAT 
               
               
                   
                   
                 TTACACACTGGCGAATGTAATCGTAAAGTTCCCGGCAGAGGAAGAATTCCATGGGTAGAAATTGGTGAGGCTCTTGCT 
               
               
                   
                   
                 GACATAGGTTATAACGGTAGTGTTGTTATGGAACCTTTTGTTAGAATGGGCGGAACTGTCGGATCTAATATTAAGGTT 
               
               
                   
                   
                 TGGCGTGACATTAGTAACGGTGCAGATGAGAAAATGCTGGATAGAGAAGCACAGGCCGCACTTGATTTCTCCAGATAT 
               
               
                   
                   
                 GTATTAGAATGTCATAAACACTCC CTCGAG   
               
               
                   
                   
                 Underlined zones are the slight modifications, in comparison with SEQ ID NO: 3 
               
               
                   
                   
                 (insertion for the restriction sites for NdeI/XhoI and the mutation T558C 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 5, 
                 MKHGIYYAYWEQEWEADYKYYIEKVAKLGFDILEIAASPLPFYSDIQINELKACAHGNGITLTVGHGPSAEQNLSSPD 
               
               
                   
                 which corresponds 
                 PDIRKNAKAFYTDLLKRLYKLDVHLIGGALYSYWPIDYTKTIDKKGDWERSVESVREVAKVAEACGVDFCLEVLNRFE 
               
               
                   
                 to the sequence of 
                 NYLINTAQEGVDFVKQVDHNNVKVMLDTFHMNIEEDSIGGAIRTAGSYLGHLHTSECNRKVPGRGRIPWVEIGEALAD 
               
               
                   
                 SEQ ID NO: 2 
                 IGYNGSVVMEPFVRMGGTVGSNIKVWRDISNGADEKMLDREAQAALDFSRYVLECHKHS 
               
               
                   
                 (having a serine 
                   
               
               
                   
                 residue at 
                   
               
               
                   
                 position 211) of 
                   
               
               
                   
                 PCT/EP2014/068628 
                   
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 6, 
                 MKHGIYYAYWEQEWEADYKYYIEKVAKLGFDILEIAASPLPFYSDIQINELKACAHGNGITLTVGHGPSAEQNLSSPD 
               
               
                   
                 which corresponds 
                 PDIRKNAKAFYTDLLKRLYKLDVHLIGGALYSYWPIDYTKTIDKKGDWERSVESVREVAKVAEACGVDFCLEVLNRFE 
               
               
                   
                 to the sequence of 
                 NYLINTAQEGVDFVKQVDHNNVKVMLDTFHMNIEEDSIGGAIRTAGSYLGHLHTSECNRKVPGRGRIPWVEIGEALAD 
               
               
                   
                 SEQ ID NO: 4 
                 IGYNGSVVMEPFVRMGGTVGSNIKVWRDISNGADEKMLDREAQAALDFSRYVLECHKHS 
               
               
                   
                 (having a serine 
                   
               
               
                   
                 residue at 
                   
               
               
                   
                 position 211) of 
                   
               
               
                   
                 PCT/EP2014/068628 
                   
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 7, 
                 MKHGIYYAYWEQEWEADYKYYIEKVAKLGFDILEIAASPLPFYSDNQINELKACARGNGITLTVGHGPSAEQNLSSPD 
               
               
                   
                 which corresponds 
                 PYIRKNAKAFYTDLLKRLYKLDVHLIGGAIYSYWPVDYTKTIDKKGDWERSVESVREVAQVAEACGVDFCLEVLNRFE 
               
               
                   
                 to the sequence of 
                 NYLINTAQEGVDFVKQVGHDNVKVMLDTFHMNIEEDSIGGAIRTAGSYLGHLHTSECNRKVPGKGRIPWIEIGEALAD 
               
               
                   
                 SEQ ID NO: 5 
                 IGYNGSVVMEPFVRMGGTVGSNIKVWRDISNGADEEKLDREAQAALNFSRYVLGNRKL 
               
               
                   
                 (having a serine 
                   
               
               
                   
                 residue at 
                   
               
               
                   
                 position 211) of 
                   
               
               
                   
                 PCT/EP2014/068628 
                   
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 8, 
                 MKHGIYYAYWEQEWAADYKRYVEKAAKLGFDILEVGAAPLPDYSAQEVKELKKCADDNGIQLTAGYGPAFNHNMGSSD 
               
               
                   
                 which corresponds 
                 PKIREEALQWYKRLFEVMAGLDIHLIGGALYSYWPVDFATANKEEDWKHSVEGMQILAPIASQYGINLGMEVLNRFES 
               
               
                   
                 to the sequence of 
                 HILNTSEEGVKFVTEVGMDNVKVMLDTFHMNIEESSIGDAIRHAGKLLGHFHTSECNRMVPGKGRTPWREIGDALREI 
               
               
                   
                 SEQ ID NO: 6 
                 EYDGTVVMEPFVRMGGQVGSDIKVWRDISKGAGEDRLDEDARRAVEFQRYMLEWK 
               
               
                   
                 (having a serine 
                   
               
               
                   
                 residue at 
                   
               
               
                   
                 position 211) of 
                   
               
               
                   
                 PCT/EP2014/068628 
                   
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 9, 
                 MKHGIYYSYWEHEWSAKFGPYIEKVAKLGFDIIEVAAHHINEYSDAELATIRKSAKDNGIILTAGIGPSKTKNLSSED 
               
               
                   
                 which corresponds 
                 AAVRAAGKAFFERTLSNVAKLDIHTIGGALHSYWPIDYSQPVDKAGDYARGVEGINGIADFANDLGINLCIEVLNRFE 
               
               
                   
                 to the sequence of 
                 NHVLNTAAEGVAFVKDVGKNNVKVMLDTFHMNIEEDSFGDAIRTAGPLLGHFHTSESNRRVPGKGRMPWHEIGLALRD 
               
               
                   
                 SEQ ID NO: 7 
                 INYTGAVIMEPFVKTGGTIGSDIKVWRDLSGGADIAKMDEDARNALAFSRFVLG 
               
               
                   
                 (having a serine 
                   
               
               
                   
                 residue at 
                   
               
               
                   
                 position 211) of 
                   
               
               
                   
                 PCT/EP2014/068628 
                   
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 10, 
                 MKYGIYYAYWEKEWNGDYKYYIDKISKLGFDILEISCGAFSDYYTKDQELIDIGKYAKEKGVTLTAGYGPHFNESLSS 
               
               
                   
                 which corresponds 
                 SEPNTQKQAISFWKETLRKLKLMDIHIVGGALYGYWPVDYSKPFDKKRDLENSIKNMKIISQYAEEYDIMMGMEVLNR 
               
               
                   
                 to the sequence of 
                 FEGYMLNTCDEALAYVEEVGSSNVGVMLDTFHMNIEEDNIAAAIRKAGDRLYHFHISEGNRKVPGKGMLPWNEIGQAL 
               
               
                   
                 SEQ ID NO: 8 
                 RDINYQHAAVMEPFVMQGGTVGHDIKIWRDIIGNCSEVTLDMDAQSALHFVKHVFEV 
               
               
                   
                 (having a serine 
                   
               
               
                   
                 residue at 
                   
               
               
                   
                 position 211) of 
                   
               
               
                   
                 PCT/EP2014/068628 
                   
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 11, 
                 MRYFKEEVAGMKYGIYFAYWTKEWFADYKKYMDKVSALGFDVLEISCAALRDVYTTKEQLIELREYAKEKGLVLTAGY 
               
               
                   
                 which corresponds 
                 GPTKAENLCSEDPEAVRRAMTFFKDLLPKLQLMDIHILGGGLYSYWPVDFTINNDKQGDRARAVRNLRELSKTAEECD 
               
               
                   
                 to the sequence of 
                 VVLGMEVLNRYEGYILNTCEEAIDFVDEIGSSHVKIMLDTFHMNIEETNMADAIRKAGDRLGHLHLSEQNRLVPGKGS 
               
               
                   
                 SEQ ID NO: 9 
                 LPWAEIGQALRDINYQGAAVMEPFVMQGGTIGSEIKVWRDMVPDLSEEALDRDAKGALEFCRHVFGI 
               
               
                   
                 (having a serine 
                   
               
               
                   
                 residue at 
                   
               
               
                   
                 position 211) of 
                   
               
               
                   
                 PCT/EP2014/068628 
                   
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 12, 
                 MNKVGMFYTYWSTEWMVDFPATAKRIAGLGFDLMEISLGEFHNLSDAKKRELKAVADDLGLTVMCCIGLKSEYDFASP 
               
               
                   
                 which corresponds 
                 DKSVRDAGTEYVKRLLDDCHLLGAPVFAGLTFCAWPQSPPLDMKDKRPYVDRAIESVRRVIKVAEDYGIIYALEVVNR 
               
               
                   
                 to the sequence of 
                 FEQWLCNDAKEAIAFADAVDSPACKVQLDTFHMNIEETSFRDAILACKGKMGHFHLSEANRLPPGEGRLPWDEIFGAL 
               
               
                   
                 SEQ ID NO: 10 
                 KEIGYDGTIVMEPFMRKGGSVSRAVGVWRDMSNGATDEEMDERARRSLQFVRDKLA 
               
               
                   
                 (having a serine 
                   
               
               
                   
                 residue at 
                   
               
               
                   
                 position 211) of 
                   
               
               
                   
                 PCT/EP2014/068628 
                   
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 13, 
                 MKNPVGIISMQFIRPFTSESLHFLKKSRALGFDFIELLVPEPEDGLDAAEVRRICEGEGLGLVLAARVNLQRSIASEE 
               
               
                   
                 which corresponds 
                 AAARAGGRDYLKYCIEAAEALGATIVGGPLYGEPLVFAGRPPFPWTAEQIATRAARTVEGLAEVAPLAASAGKVFGLE 
               
               
                   
                 to thes equence of 
                 PLNRFETDIVNTTAQAIEVVDAVGSPGLGVMLDTFHMNMEERSIPDAIRATGARLVHFQANENHRGFPGTGTMDWTAI 
               
               
                   
                 SEQ ID NO: 11 
                 ARALGQAGYAGPVSLEPFRRDDERVALPIAHWRAPHEDEDEKLRAGLGLIRSAITLAEVTH 
               
               
                   
                 (having a serine 
                   
               
               
                   
                 residue at 
                   
               
               
                   
                 position 211) of 
                   
               
               
                   
                 PCT/EP2014/068628 
                   
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 14, 
                 CTTAAGGAACGTACAGACGGCTTAAAAGCCTTTAAAAACGTTTTTAAGGGGTTTGTAGACAAGGTAAAGGATAAAACA 
               
               
                   
                 plasmid pR1 
                 GCACAATTCCAAGAAAAACACGATTTAGAACCTAAAAAGAACGAATTTGAACTAACTCATAACCGAGAGGTAAAAAAA 
               
               
                   
                   
                 GAACGAAGTCGAGATCAGGGAATGAGTTTATAAAATAAAAAAAGCACCTGAAAAGGTGTCTTTTTTTGATGGTTTTGA 
               
               
                   
                   
                 ACTTGTTCTTTCTTATCTTGATACATATAGAAATAACGTCATTTTTATTTTAGTTGCTGAAAGGTGCGTTGAAGTGTT 
               
               
                   
                   
                 GGTATGTATGTGTTTTAAAGTATTGAAAACCCTTAAAATTGGTTGCACAGAAAAACCCCATCTGTTAAAGTTATAAGT 
               
               
                   
                   
                 GACTAAACAAATAACTAAATAGATGGGGGTTTCTTTTAATATTATGTGTCCTAATAGTAGCATTTATTCAGATGAAAA 
               
               
                   
                   
                 ATCAAGGGTTTTAGTGGACAAGACAAAAAGTGGAAAAGTGAGACCATGGAGAGAAAAGAAAATCGCTAATGTTGATTA 
               
               
                   
                   
                 CTTTGAACTTCTGCATATTCTTGAATTTAAAAAGGCTGAAAGAGTAAAAGATTGTGCTGAAATATTAGAGTATAAACA 
               
               
                   
                   
                 AAATCGTGAAACAGGCGAAAGAAAGTTGTATCGAGTGTGGTTTTGTAAATCCAGGCTTTGTCCAATGTGCAACTGGAG 
               
               
                   
                   
                 GAGAGCAATGAAACATGGCATTCAGTCACAAAAGGTTGTTGCTGAAGTTATTAAACAAAAGCCAACAGTTCGTTGGTT 
               
               
                   
                   
                 GTTTCTCACATTAACAGTTAAAAATGTTTATGATGGCGAAGAATTAAATAAGAGTTTGTCAGATATGGCTCAAGGATT 
               
               
                   
                   
                 TCGCCGAATGATGCAATATAAAAAAATTAATAAAAATCTTGTTGGTTTTATGCGTGCAACGGAAGTGACAATAAATAA 
               
               
                   
                   
                 TAAAGATAATTCTTATAATCAGCACATGCATGTATTGGTATGTGTGGAACCAACTTATTTTAAGAATACAGAAAACTA 
               
               
                   
                   
                 CGTGAATCAAAAACAATGGATTCAATTTTGGAAAAAGGCAATGAAATTAGACTATGATCCAAATGTAAAAGTTCAAAT 
               
               
                   
                   
                 GATTCGACCGAAAAATAAATATAAATCGGATATACAATCGGCAATTGACGAAACTGCAAAATATCCTGTAAAGGATAC 
               
               
                   
                   
                 GGATTTTATGACCGATGATGAAGAAAAGAATTTGAAACGTTTGTCTGATTTGGAGGAAGGTTTACACCGTAAAAGGTT 
               
               
                   
                   
                 AATCTCCTATGGTGGTTTGTTAAAAGAAATACATAAAAAATTAAACCTTGATGACACAGAAGAAGGCGATTTGATTCA 
               
               
                   
                   
                 TACAGATGATGACGAAAAAGCCGATGAAGATGGATTTTCTATTATTGCAATGTGGAATTGGGAACGGAAAAATTATTT 
               
               
                   
                   
                 TATTAAAGAGTAGTTCAACAAACGGGCCAGTTTGTTGAAGATTAGATGCTATAATTGTTATTAAAAGGATTGAAGGAT 
               
               
                   
                   
                 GCTTAGGAAGACGAGTTATTAATAGCTGAATAAGAACGGTGCTCTCCAAATATTCTTATTTAGAAAAGCAAATCTAAA 
               
               
                   
                   
                 ATTATCTGAAAAGGGAAGATCTTTCTAAAGAGGAAATGGTGACAGTAGCGAAAAGCATGCAGGGACAATCATCGAAAT 
               
               
                   
                   
                 AACCGCCAAAGGCCAAACATGATTTGGCCTTTTTTTCGTTAGACATCGTTTCCCTTTAGCCTTTAATTTTAGTATGAT 
               
               
                   
                   
                 ATGTAAATGATATTGAATAAAAGCTAGGAAGTGTCGTAATGAGCACAAAACCTTTTTACAGAGATACGTGGGCGGAAA 
               
               
                   
                   
                 TTGACTTGTCCGCGATAAAGGAAAATGTCAGCAATATGAAAAAACATATCGGTGAACATGTCCACTTGATGGCAGTTG 
               
               
                   
                   
                 TGAAAGCAAACGCCTACGGGCATGGTGATGCAGAAACAGCAAAGGCTGCTCTTGACGCAGGTGCTTCATGCTTGGCCG 
               
               
                   
                   
                 TGGCCATTTTGGATGAAGCGATTTCACTGCGCAAAAAGGGATTGAAGGCGCCTATATTGGTGCTTGGCGCGGTTCCCC 
               
               
                   
                   
                 CGGAGTATGTGGCAATCGCTGCTGAGTATGACGTGACCTTAACAGGTTATTCTGTTGAATGGCTTCAGGAGGCAGCCC 
               
               
                   
                   
                 GCCACACGAAAAAAGGTTCTCTTCATTTTCATCTGAAGGTCGATACGGGGATGAACAGACTTGGTGTAAAAACAGAGG 
               
               
                   
                   
                 AAGAAGTTCAGAACGTGATGGCAATTCTTGACCGCAACCCTCGTTTAAAGTGCAAAGGGGTATTTACCCATTTTGCGA 
               
               
                   
                   
                 CAGCGGATGAAAAAGAAAGAGGCTATTTCTTAATGCAGTTTGAGCGCTTTAAAGAGCTGATTGCTCCGCTGCCGTTAA 
               
               
                   
                   
                 AGAATCTAATGGTCCACTGCGCGAACAGCGCCGCTGGACTCCGGCTGAAAAAAGGCTTTTTTAATGCAGTCAGATTCG 
               
               
                   
                   
                 GCATCGGCATGTATGGCCTTCGCCCGTCTGCTGACATGTCGGACGAGATACCGTTTCAGCTGCGTCCGGCATTTACCC 
               
               
                   
                   
                 TGCATTCGACACTGTCACATGTCAAACTGATCAGAAAAGGCGAGAGCGTCAGCTACGGAGCCGAGTACACAGCGGAAA 
               
               
                   
                   
                 AAGACACATGGATCGGGACGGTGCCTGTAGGCTATGCGGACGGCTGGCTCCGAAAATTGAAAGGGACCGACATCCTTG 
               
               
                   
                   
                 TGAAGGGAAAACGCCTGAAAATTGCCGGCCGAATTTGCATGGACCAATTTATGGTGGAGCTGGATCAGGAATATCCGC 
               
               
                   
                   
                 CGGGCACAAAAGTCACATTAATAGGCCGGCAGGGGGATGAATATATTTCCATGGATGAGATTGCAGGAAGGCTCGAAA 
               
               
                   
                   
                 CCATTAACTATGAGGTGGCCTGTACAATAAGTTCCCGTGTTCCCCGTATGTTTTTGGAAAATGGGAGTATAATGGAAG 
               
               
                   
                   
                 TAAGAAATCCTTTATTGCAGGTAAATATAAGCAATTAACTTACCTAAATGGAGAATTCAATCTATTATTAATCTGTTC 
               
               
                   
                   
                 AGCAATCGGGCGCGATTGCTGAATAAAAGATACGAGAGACCTCTCTTGTATCTTTTTTATTTTGAGTGGTTTTGTCCG 
               
               
                   
                   
                 TTACACTAGAAAACCGAAAGACAATAAAAATTTTATTCTTGCTGAGTCTGGCTTTCGGTAAGCTAGACAAAACGGACA 
               
               
                   
                   
                 AAATAAAAATTGGCAAGGGTTTAAAGGTGGAGATTTTTTGAGTGATCTTCTCAAAAAATACTACCTGTCCCTTGCTGA 
               
               
                   
                   
                 TTTTTAAACGAGCACGAGAGCAAAACCCCCCTTTGCTGAGGTGGCAGAGGGCAGGTTTTTTTGTTTCTTTTTTCTCGT 
               
               
                   
                   
                 AAAAAAAAGAAAGGTCTTAAAGGTTTTATGGTTTTGGTCGGCACTGCCGACAGCCTCGCAGAGCACACACTTTATGAA 
               
               
                   
                   
                 TATAAAGTATAGTGTGTTATACTTTACTTGGAAGTGGTTGCCGGAAAGAGCGAAAATGCCTCACATTTGTGCCACCTA 
               
               
                   
                   
                 AAAAGGAGCGATTTACATATGAGTTATGCAGTTTGTAGAATGCAAAAAGTGAAATCATAATGATAGGTGGTATGTTTT 
               
               
                   
                   
                 CGCTTGAACTTTTAAATACAGCCATTGAACATACGGTTGATTTAATAACTGACAAACATCACCCTCTTGCTAAAGCGG 
               
               
                   
                   
                 CCAAGGACGCTGCCGCCGGGGCTGTTTGCGTTTTTGCCGTGATTTCGTGTATCATTGGTTTACTTATTTTTTTGCCAA 
               
               
                   
                   
                 AGCTGTAATGGCTGAAAATTCTTACATTTATATTTACATTTTTAGAAATGGGCGTGAAAAAAAGCGCGCGATTATGTA 
               
               
                   
                   
                 AAATATAAAGTGATAGCGGTACCATTATAGGTAAGAGAGGAATGTACACATGAAACATGGTATATACTACGCATATTG 
               
               
                   
                   
                 GGAACAAGAATGGGAAGCTGATTACAAATACTATATTGAGAAGGTTGCAAAGCTTGGTTTTGATATTCTAGAGATTGC 
               
               
                   
                   
                 AGCTTCACCGCTACCTTTTTACAGTGACATTCAGATTAATGAGCTCAAGGCATGTGCCCATGGCAATGGAATTACACT 
               
               
                   
                   
                 TACGGTAGGCCATGGGCCTAGTGCAGAACAAAACCTGTCTTCTCCCGACCCCGATATTCGCAAAAATGCTAAAGCTTT 
               
               
                   
                   
                 TTATACCGATTTACTCAAACGACTTTACAAGCTGGATGTACATTTGATAGGTGGGGCTTTATATTCTTATTGGCCGAT 
               
               
                   
                   
                 AGATTACACAAAGACAATTGATAAAAAAGGCGATTGGGAACGCAGCGTTGAAAGTGTTCGAGAAGTTGCTAAGGTGGC 
               
               
                   
                   
                 CGAAGCCTGTGGAGTGGATTTCTGCCTAGAGGTTCTTAATAGATTTGAGAATTATTTAATTAACACAGCACAAGAGGG 
               
               
                   
                   
                 TGTAGATTTTGTAAAACAGGTTGACCATAACAATGTAAAGGTAATGCTTGATACCTTCCACATGAATATTGAGGAAGA 
               
               
                   
                   
                 TAGTATCGGAGGTGCAATCAGGACTGCGGGCTCTTACTTGGGACATTTACACACTGGCGAATGTAATCGTAAAGTTCC 
               
               
                   
                   
                 CGGCAGAGGAAGAATTCCATGGGTAGAAATTGGTGAGGCTCTTGCTGACATAGGTTATAACGGTAGTGTTGTTATGGA 
               
               
                   
                   
                 ACCTTTTGTTAGAATGGGCGGAACTGTCGGATCTAATATTAAGGTTTGGCGTGACATTAGTAACGGTGCAGATGAGAA 
               
               
                   
                   
                 AATGCTGGATAGAGAAGCACAGGCCGCACTTGATTTCTCCAGATATGTATTAGAATGTCATAAACACTCCTAAGAATT 
               
               
                   
                   
                 C 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 15, 
                 CTTAAGGAACGTACAGACGGCTTAAAAGCCTTTAAAAACGTTTTTAAGGGGTTTGTAGACAAGGTAAAGGATAAAACA 
               
               
                   
                 plasmid pR2 
                 GCACAATTCCAAGAAAAACACGATTTAGAACCTAAAAAGAACGAATTTGAACTAACTCATAACCGAGAGGTAAAAAAA 
               
               
                   
                   
                 GAACGAAGTCGAGATCAGGGAATGAGTTTATAAAATAAAAAAAGCACCTGAAAAGGTGTCTTTTTTTGATGGTTTTGA 
               
               
                   
                   
                 ACTTGTTCTTTCTTATCTTGATACATATAGAAATAACGTCATTTTTATTTTAGTTGCTGAAAGGTGCGTTGAAGTGTT 
               
               
                   
                   
                 GGTATGTATGTGTTTTAAAGTATTGAAAACCCTTAAAATTGGTTGCACAGAAAAACCCCATCTGTTAAAGTTATAAGT 
               
               
                   
                   
                 GACTAAACAAATAACTAAATAGATGGGGGTTTCTTTTAATATTATGTGTCCTAATAGTAGCATTTATTCAGATGAAAA 
               
               
                   
                   
                 ATCAAGGGTTTTAGTGGACAAGACAAAAAGTGGAAAAGTGAGACCATGGAGAGAAAAGAAAATCGCTAATGTTGATTA 
               
               
                   
                   
                 CTTTGAACTTCTGCATATTCTTGAATTTAAAAAGGCTGAAAGAGTAAAAGATTGTGCTGAAATATTAGAGTATAAACA 
               
               
                   
                   
                 AAATCGTGAAACAGGCGAAAGAAAGTTGTATCGAGTGTGGTTTTGTAAATCCAGGCTTTGTCCAATGTGCAACTGGAG 
               
               
                   
                   
                 GAGAGCAATGAAACATGGCATTCAGTCACAAAAGGTTGTTGCTGAAGTTATTAAACAAAAGCCAACAGTTCGTTGGTT 
               
               
                   
                   
                 GTTTCTCACATTAACAGTTAAAAATGTTTATGATGGCGAAGAATTAAATAAGAGTTTGTCAGATATGGCTCAAGGATT 
               
               
                   
                   
                 TCGCCGAATGATGCAATATAAAAAAATTAATAAAAATCTTGTTGGTTTTATGCGTGCAACGGAAGTGACAATAAATAA 
               
               
                   
                   
                 TAAAGATAATTCTTATAATCAGCACATGCATGTATTGGTATGTGTGGAACCAACTTATTTTAAGAATACAGAAAACTA 
               
               
                   
                   
                 CGTGAATCAAAAACAATGGATTCAATTTTGGAAAAAGGCAATGAAATTAGACTATGATCCAAATGTAAAAGTTCAAAT 
               
               
                   
                   
                 GATTCGACCGAAAAATAAATATAAATCGGATATACAATCGGCAATTGACGAAACTGCAAAATATCCTGTAAAGGATAC 
               
               
                   
                   
                 GGATTTTATGACCGATGATGAAGAAAAGAATTTGAAACGTTTGTCTGATTTGGAGGAAGGTTTACACCGTAAAAGGTT 
               
               
                   
                   
                 AATCTCCTATGGTGGTTTGTTAAAAGAAATACATAAAAAATTAAACCTTGATGACACAGAAGAAGGCGATTTGATTCA 
               
               
                   
                   
                 TACAGATGATGACGAAAAAGCCGATGAAGATGGATTTTCTATTATTGCAATGTGGAATTGGGAACGGAAAAATTATTT 
               
               
                   
                   
                 TATTAAAGAGTAGTTCAACAAACGGGCCAGTTTGTTGAAGATTAGATGCTATAATTGTTATTAAAAGGATTGAAGGAT 
               
               
                   
                   
                 GCTTAGGAAGACGAGTTATTAATAGCTGAATAAGAACGGTGCTCTCCAAATATTCTTATTTAGAAAAGCAAATCTAAA 
               
               
                   
                   
                 ATTATCTGAAAAGGGAAGATCTTTCTAAAGAGGAAATGGTGACAGTAGCGAAAAGCATGCAGGGACAATCATCGAAAT 
               
               
                   
                   
                 AACCGCCAAAGGCCAAACATGATTTGGCCTTTTTTTCGTTAGACATCGTTTCCCTTTAGCCTTTAATTTTAGTATGAT 
               
               
                   
                   
                 ATGTAAATGATATTGAATAAAAGCTAGGAAGTGTCGTAATGAGCACAAAACCTTTTTACAGAGATACGTGGGCGGAAA 
               
               
                   
                   
                 TTGACTTGTCCGCGATAAAGGAAAATGTCAGCAATATGAAAAAACATATCGGTGAACATGTCCACTTGATGGCAGTTG 
               
               
                   
                   
                 TGAAAGCAAACGCCTACGGGCATGGTGATGCAGAAACAGCAAAGGCTGCTCTTGACGCAGGTGCTTCATGCTTGGCCG 
               
               
                   
                   
                 TGGCCATTTTGGATGAAGCGATTTCACTGCGCAAAAAGGGATTGAAGGCGCCTATATTGGTGCTTGGCGCGGTTCCCC 
               
               
                   
                   
                 CGGAGTATGTGGCAATCGCTGCTGAGTATGACGTGACCTTAACAGGTTATTCTGTTGAATGGCTTCAGGAGGCAGCCC 
               
               
                   
                   
                 GCCACACGAAAAAAGGTTCTCTTCATTTTCATCTGAAGGTCGATACGGGGATGAACAGACTTGGTGTAAAAACAGAGG 
               
               
                   
                   
                 AAGAAGTTCAGAACGTGATGGCAATTCTTGACCGCAACCCTCGTTTAAAGTGCAAAGGGGTATTTACCCATTTTGCGA 
               
               
                   
                   
                 CAGCGGATGAAAAAGAAAGAGGCTATTTCTTAATGCAGTTTGAGCGCTTTAAAGAGCTGATTGCTCCGCTGCCGTTAA 
               
               
                   
                   
                 AGAATCTAATGGTCCACTGCGCGAACAGCGCCGCTGGACTCCGGCTGAAAAAAGGCTTTTTTAATGCAGTCAGATTCG 
               
               
                   
                   
                 GCATCGGCATGTATGGCCTTCGCCCGTCTGCTGACATGTCGGACGAGATACCGTTTCAGCTGCGTCCGGCATTTACCC 
               
               
                   
                   
                 TGCATTCGACACTGTCACATGTCAAACTGATCAGAAAAGGCGAGAGCGTCAGCTACGGAGCCGAGTACACAGCGGAAA 
               
               
                   
                   
                 AAGACACATGGATCGGGACGGTGCCTGTAGGCTATGCGGACGGCTGGCTCCGAAAATTGAAAGGGACCGACATCCTTG 
               
               
                   
                   
                 TGAAGGGAAAACGCCTGAAAATTGCCGGCCGAATTTGCATGGACCAATTTATGGTGGAGCTGGATCAGGAATATCCGC 
               
               
                   
                   
                 CGGGCACAAAAGTCACATTAATAGGCCGGCAGGGGGATGAATATATTTCCATGGATGAGATTGCAGGAAGGCTCGAAA 
               
               
                   
                   
                 CCATTAACTATGAGGTGGCCTGTACAATAAGTTCCCGTGTTCCCCGTATGTTTTTGGAAAATGGGAGTATAATGGAAG 
               
               
                   
                   
                 TAAGAAATCCTTTATTGCAGGTAAATATAAGCAATTAACTTACCTAAATGGAGAATTCAATCTATTATTAATCTGTTC 
               
               
                   
                   
                 AGCAATCGGGCGCGATTGCTGAATAAAAGATACGAGAGACCTCTCTTGTATCTTTTTTATTTTGAGTGGTTTTGTCCG 
               
               
                   
                   
                 TTACACTAGAAAACCGAAAGACAATAAAAATTTTATTCTTGCTGAGTCTGGCTTTCGGTAAGCTAGACAAAACGGACA 
               
               
                   
                   
                 AAATAAAAATTGGCAAGGGTTTAAAGGTGGAGATTTTTTGAGTGATCTTCTCAAAAAATACTACCTGTCCCTTGCTGA 
               
               
                   
                   
                 TTTTTAAACGAGCACGAGAGCAAAACCCCCCTTTGCTGAGGTGGCAGAGGGCAGGTTTTTTTGTTTCTTTTTTCTCGT 
               
               
                   
                   
                 AAAAAAAAGAAAGGTCTTAAAGGTTTTATGGTTTTGGTCGGCACTGCCGACAGCCTCGCAGAGCACACACTTTATGAA 
               
               
                   
                   
                 TATAAAGTATAGTGTGTTATACTTTACTTGGAAGTGGTTGCCGGAAAGAGCGAAAATGCCTCACATTTGTGCCACCTA 
               
               
                   
                   
                 AAAAGGAGCGATTTACATATGAGTTATGCAGTTTGTAGAATGCAAAAAGTGAAATCATAATGATAGGTGGTATGTTTT 
               
               
                   
                   
                 CGCTTGAACTTTTAAATACAGCCATTGAACATACGGTTGATTTAATAACTGACAAACATCACCCTCTTGCTAAAGCGG 
               
               
                   
                   
                 CCAAGGACGCTGCCGCCGGGGCTGTTTGCGTTTTTGCCGTGATTTCGTGTATCATTGGTTTACTTATTTTTTTGCCAA 
               
               
                   
                   
                 AGCTGTAATGGCTGAAAATTCTTACATTTATATTTACATTTTTAGAAATGGGCGTGAAAAAAAGCGCGCGATTATGTA 
               
               
                   
                   
                 AAATATAAAGTGATAGCGGTACCATTATAGGTAGAAAGGAGGATTACATATGAAACATGGTATATACTACGCATATTG 
               
               
                   
                   
                 GGAACAAGAATGGGAAGCTGATTACAAATACTATATTGAGAAGGTTGCAAAGCTTGGTTTTGATATTCTAGAGATTGC 
               
               
                   
                   
                 AGCTTCACCGCTACCTTTTTACAGTGACATTCAGATTAATGAGCTCAAGGCATGTGCCCATGGCAATGGAATTACACT 
               
               
                   
                   
                 TACGGTAGGCCATGGGCCTAGTGCAGAACAAAACCTGTCTTCTCCCGACCCCGATATTCGCAAAAATGCTAAAGCTTT 
               
               
                   
                   
                 TTATACCGATTTACTCAAACGACTTTACAAGCTGGATGTACATTTGATAGGTGGGGCTTTATATTCTTATTGGCCGAT 
               
               
                   
                   
                 AGATTACACAAAGACAATTGATAAAAAAGGCGATTGGGAACGCAGCGTTGAAAGTGTTCGAGAAGTTGCTAAGGTGGC 
               
               
                   
                   
                 CGAAGCCTGTGGAGTGGATTTCTGCCTAGAGGTTCTTAATAGATTTGAGAATTATTTAATTAACACAGCACAAGAGGG 
               
               
                   
                   
                 TGTAGATTTTGTAAAACAGGTTGACCATAACAATGTAAAGGTAATGCTTGATACCTTCCACATGAATATTGAGGAAGA 
               
               
                   
                   
                 TAGTATCGGAGGTGCAATCAGGACTGCGGGCTCTTACTTGGGACATTTACACACTGGCGAATGTAATCGTAAAGTTCC 
               
               
                   
                   
                 CGGCAGAGGAAGAATTCCATGGGTAGAAATTGGTGAGGCTCTTGCTGACATAGGTTATAACGGTAGTGTTGTTATGGA 
               
               
                   
                   
                 ACCTTTTGTTAGAATGGGCGGAACTGTCGGATCTAATATTAAGGTTTGGCGTGACATTAGTAACGGTGCAGATGAGAA 
               
               
                   
                   
                 AATGCTGGATAGAGAAGCACAGGCCGCACTTGATTTCTCCAGATATGTATTAGAATGTCATAAACACTCCTAAGAATT 
               
               
                   
                   
                 C 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 16, 
                 CTTAAGGAACGTACAGACGGCTTAAAAGCCTTTAAAAACGTTTTTAAGGGGTTTGTAGACAAGGTAAAGGATAAAACA 
               
               
                   
                 plasmid pR3 
                 GCACAATTCCAAGAAAAACACGATTTAGAACCTAAAAAGAACGAATTTGAACTAACTCATAACCGAGAGGTAAAAAAA 
               
               
                   
                   
                 GAACGAAGTCGAGATCAGGGAATGAGTTTATAAAATAAAAAAAGCACCTGAAAAGGTGTCTTTTTTTGATGGTTTTGA 
               
               
                   
                   
                 ACTTGTTCTTTCTTATCTTGATACATATAGAAATAACGTCATTTTTATTTTAGTTGCTGAAAGGTGCGTTGAAGTGTT 
               
               
                   
                   
                 GGTATGTATGTGTTTTAAAGTATTGAAAACCCTTAAAATTGGTTGCACAGAAAAACCCCATCTGTTAAAGTTATAAGT 
               
               
                   
                   
                 GACTAAACAAATAACTAAATAGATGGGGGTTTCTTTTAATATTATGTGTCCTAATAGTAGCATTTATTCAGATGAAAA 
               
               
                   
                   
                 ATCAAGGGTTTTAGTGGACAAGACAAAAAGTGGAAAAGTGAGACCATGGAGAGAAAAGAAAATCGCTAATGTTGATTA 
               
               
                   
                   
                 CTTTGAACTTCTGCATATTCTTGAATTTAAAAAGGCTGAAAGAGTAAAAGATTGTGCTGAAATATTAGAGTATAAACA 
               
               
                   
                   
                 AAATCGTGAAACAGGCGAAAGAAAGTTGTATCGAGTGTGGTTTTGTAAATCCAGGCTTTGTCCAATGTGCAACTGGAG 
               
               
                   
                   
                 GAGAGCAATGAAACATGGCATTCAGTCACAAAAGGTTGTTGCTGAAGTTATTAAACAAAAGCCAACAGTTCGTTGGTT 
               
               
                   
                   
                 GTTTCTCACATTAACAGTTAAAAATGTTTATGATGGCGAAGAATTAAATAAGAGTTTGTCAGATATGGCTCAAGGATT 
               
               
                   
                   
                 TCGCCGAATGATGCAATATAAAAAAATTAATAAAAATCTTGTTGGTTTTATGCGTGCAACGGAAGTGACAATAAATAA 
               
               
                   
                   
                 TAAAGATAATTCTTATAATCAGCACATGCATGTATTGGTATGTGTGGAACCAACTTATTTTAAGAATACAGAAAACTA 
               
               
                   
                   
                 CGTGAATCAAAAACAATGGATTCAATTTTGGAAAAAGGCAATGAAATTAGACTATGATCCAAATGTAAAAGTTCAAAT 
               
               
                   
                   
                 GATTCGACCGAAAAATAAATATAAATCGGATATACAATCGGCAATTGACGAAACTGCAAAATATCCTGTAAAGGATAC 
               
               
                   
                   
                 GGATTTTATGACCGATGATGAAGAAAAGAATTTGAAACGTTTGTCTGATTTGGAGGAAGGTTTACACCGTAAAAGGTT 
               
               
                   
                   
                 AATCTCCTATGGTGGTTTGTTAAAAGAAATACATAAAAAATTAAACCTTGATGACACAGAAGAAGGCGATTTGATTCA 
               
               
                   
                   
                 TACAGATGATGACGAAAAAGCCGATGAAGATGGATTTTCTATTATTGCAATGTGGAATTGGGAACGGAAAAATTATTT 
               
               
                   
                   
                 TATTAAAGAGTAGTTCAACAAACGGGCCAGTTTGTTGAAGATTAGATGCTATAATTGTTATTAAAAGGATTGAAGGAT 
               
               
                   
                   
                 GCTTAGGAAGACGAGTTATTAATAGCTGAATAAGAACGGTGCTCTCCAAATATTCTTATTTAGAAAAGCAAATCTAAA 
               
               
                   
                   
                 ATTATCTGAAAAGGGAAGATCTTTCTAAAGAGGAAATGGTGACAGTAGCGAAAAGCATGCAGGGACAATCATCGAAAT 
               
               
                   
                   
                 AACCGCCAAAGGCCAAACATGATTTGGCCTTTTTTTCGTTAGACATCGTTTCCCTTTAGCCTTTAATTTTAGTATGAT 
               
               
                   
                   
                 ATGTAAATGATATTGAATAAAAGCTAGGAAGTGTCGTAATGAGCACAAAACCTTTTTACAGAGATACGTGGGCGGAAA 
               
               
                   
                   
                 TTGACTTGTCCGCGATAAAGGAAAATGTCAGCAATATGAAAAAACATATCGGTGAACATGTCCACTTGATGGCAGTTG 
               
               
                   
                   
                 TGAAAGCAAACGCCTACGGGCATGGTGATGCAGAAACAGCAAAGGCTGCTCTTGACGCAGGTGCTTCATGCTTGGCCG 
               
               
                   
                   
                 TGGCCATTTTGGATGAAGCGATTTCACTGCGCAAAAAGGGATTGAAGGCGCCTATATTGGTGCTTGGCGCGGTTCCCC 
               
               
                   
                   
                 CGGAGTATGTGGCAATCGCTGCTGAGTATGACGTGACCTTAACAGGTTATTCTGTTGAATGGCTTCAGGAGGCAGCCC 
               
               
                   
                   
                 GCCACACGAAAAAAGGTTCTCTTCATTTTCATCTGAAGGTCGATACGGGGATGAACAGACTTGGTGTAAAAACAGAGG 
               
               
                   
                   
                 AAGAAGTTCAGAACGTGATGGCAATTCTTGACCGCAACCCTCGTTTAAAGTGCAAAGGGGTATTTACCCATTTTGCGA 
               
               
                   
                   
                 CAGCGGATGAAAAAGAAAGAGGCTATTTCTTAATGCAGTTTGAGCGCTTTAAAGAGCTGATTGCTCCGCTGCCGTTAA 
               
               
                   
                   
                 AGAATCTAATGGTCCACTGCGCGAACAGCGCCGCTGGACTCCGGCTGAAAAAAGGCTTTTTTAATGCAGTCAGATTCG 
               
               
                   
                   
                 GCATCGGCATGTATGGCCTTCGCCCGTCTGCTGACATGTCGGACGAGATACCGTTTCAGCTGCGTCCGGCATTTACCC 
               
               
                   
                   
                 TGCATTCGACACTGTCACATGTCAAACTGATCAGAAAAGGCGAGAGCGTCAGCTACGGAGCCGAGTACACAGCGGAAA 
               
               
                   
                   
                 AAGACACATGGATCGGGACGGTGCCTGTAGGCTATGCGGACGGCTGGCTCCGAAAATTGAAAGGGACCGACATCCTTG 
               
               
                   
                   
                 TGAAGGGAAAACGCCTGAAAATTGCCGGCCGAATTTGCATGGACCAATTTATGGTGGAGCTGGATCAGGAATATCCGC 
               
               
                   
                   
                 CGGGCACAAAAGTCACATTAATAGGCCGGCAGGGGGATGAATATATTTCCATGGATGAGATTGCAGGAAGGCTCGAAA 
               
               
                   
                   
                 CCATTAACTATGAGGTGGCCTGTACAATAAGTTCCCGTGTTCCCCGTATGTTTTTGGAAAATGGGAGTATAATGGAAG 
               
               
                   
                   
                 TAAGAAATCCTTTATTGCAGGTAAATATAAGCAATTAACTTACCTAAATGGAGAATTCAATCTATTATTAATCTGTTC 
               
               
                   
                   
                 AGCAATCGGGCGCGATTGCTGAATAAAAGATACGAGAGACCTCTCTTGTATCTTTTTTATTTTGAGTGGTTTTGTCCG 
               
               
                   
                   
                 TTACACTAGAAAACCGAAAGACAATAAAAATTTTATTCTTGCTGAGTCTGGCTTTCGGTAAGCTAGACAAAACGGACA 
               
               
                   
                   
                 AAATAAAAATTGGCAAGGGTTTAAAGGTGGAGATTTTTTGAGTGATCTTCTCAAAAAATACTACCTGTCCCTTGCTGA 
               
               
                   
                   
                 TTTTTAAACGAGCACGAGAGCAAAACCCCCCTTTGCTGAGGTGGCAGAGGGCAGGTTTTTTTGTTTCTTTTTTCTCGT 
               
               
                   
                   
                 AAAAAAAAGAAAGGTCTTAAAGGTTTTATGGTTTTGGTCGGCACTGCCGACAGCCTCGCAGAGCACACACTTTATGAA 
               
               
                   
                   
                 TATAAAGTATAGTGTGTTATACTTTACTTGGAAGTGGTTGCCGGAAAGAGCGAAAATGCCTCACATTTGTGCCACCTA 
               
               
                   
                   
                 AAAAGGAGCGATTTACATATGAGTTATGCAGTTTGTAGAATGCAAAAAGTGAAATCATAATGATAGGTGGTATGTTTT 
               
               
                   
                   
                 CGCTTGAACTTTTAAATACAGCCATTGAACATACGGTTGATTTAATAACTGACAAACATCACCCTCTTGCTAAAGCGG 
               
               
                   
                   
                 CCAAGGACGCTGCCGCCGGGGCTGTTTGCGTTTTTGCCGTGATTTCGTGTATCATTGGTTTACTTATTTTTTTGCCAA 
               
               
                   
                   
                 AGCTGTAATGGCTGAAAATTCTTACATTTATATTTACATTTTTAGAAATGGGCGTGAAAAAAAGCGCGCGATTATGTA 
               
               
                   
                   
                 AAATATAAAGTGATAGCGGTACCATTATAGGTAGAAAGGAGGATTCGAAATGAAACATGGTATATACTACGCATATTG 
               
               
                   
                   
                 GGAACAAGAATGGGAAGCTGATTACAAATACTATATTGAGAAGGTTGCAAAGCTTGGTTTTGATATTCTAGAGATTGC 
               
               
                   
                   
                 AGCTTCACCGCTACCTTTTTACAGTGACATTCAGATTAATGAGCTCAAGGCATGTGCCCATGGCAATGGAATTACACT 
               
               
                   
                   
                 TACGGTAGGCCATGGGCCTAGTGCAGAACAAAACCTGTCTTCTCCCGACCCCGATATTCGCAAAAATGCTAAAGCTTT 
               
               
                   
                   
                 TTATACCGATTTACTCAAACGACTTTACAAGCTGGATGTACATTTGATAGGTGGGGCTTTATATTCTTATTGGCCGAT 
               
               
                   
                   
                 AGATTACACAAAGACAATTGATAAAAAAGGCGATTGGGAACGCAGCGTTGAAAGTGTTCGAGAAGTTGCTAAGGTGGC 
               
               
                   
                   
                 CGAAGCCTGTGGAGTGGATTTCTGCCTAGAGGTTCTTAATAGATTTGAGAATTATTTAATTAACACAGCACAAGAGGG 
               
               
                   
                   
                 TGTAGATTTTGTAAAACAGGTTGACCATAACAATGTAAAGGTAATGCTTGATACCTTCCACATGAATATTGAGGAAGA 
               
               
                   
                   
                 TAGTATCGGAGGTGCAATCAGGACTGCGGGCTCTTACTTGGGACATTTACACACTGGCGAATGTAATCGTAAAGTTCC 
               
               
                   
                   
                 CGGCAGAGGAAGAATTCCATGGGTAGAAATTGGTGAGGCTCTTGCTGACATAGGTTATAACGGTAGTGTTGTTATGGA 
               
               
                   
                   
                 ACCTTTTGTTAGAATGGGCGGAACTGTCGGATCTAATATTAAGGTTTGGCGTGACATTAGTAACGGTGCAGATGAGAA 
               
               
                   
                   
                 AATGCTGGATAGAGAAGCACAGGCCGCACTTGATTTCTCCAGATATGTATTAGAATGTCATAAACACTCCTAAGAATT 
               
               
                   
                   
                 C 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 17, 
                 ATGAGCACAAAACCTTTTTACAGAGATACGTGGGCGGAAATTGACTTGTCCGCGATAAAGGAAAATGTCAGCAATATG 
               
               
                   
                 alrA gene 
                 AAAAAACATATCGGTGAACATGTCCACTTGATGGCAGTTGTGAAAGCAAACGCCTACGGGCATGGTGATGCAGAAACA 
               
               
                   
                   
                 GCAAAGGCTGCTCTTGACGCAGGTGCTTCATGCTTGGCCGTGGCCATTTTGGATGAAGCGATTTCACTGCGCAAAAAG 
               
               
                   
                   
                 GGATTGAAGGCGCCTATATTGGTGCTTGGCGCGGTTCCCCCGGAGTATGTGGCAATCGCTGCTGAGTATGACGTGACC 
               
               
                   
                   
                 TTAACAGGTTATTCTGTTGAATGGCTTCAGGAGGCAGCCCGCCACACGAAAAAAGGTTCTCTTCATTTTCATCTGAAG 
               
               
                   
                   
                 GTCGATACGGGGATGAACAGACTTGGTGTAAAAACAGAGGAAGAAGTTCAGAACGTGATGGCAATTCTTGACCGCAAC 
               
               
                   
                   
                 CCTCGTTTAAAGTGCAAAGGGGTATTTACCCATTTTGCGACAGCGGATGAAAAAGAAAGAGGCTATTTCTTAATGCAG 
               
               
                   
                   
                 TTTGAGCGCTTTAAAGAGCTGATTGCTCCGCTGCCGTTAAAGAATCTAATGGTCCACTGCGCGAACAGCGCCGCTGGA 
               
               
                   
                   
                 CTCCGGCTGAAAAAAGGCTTTTTTAATGCAGTCAGATTCGGCATCGGCATGTATGGCCTTCGCCCGTCTGCTGACATG 
               
               
                   
                   
                 TCGGACGAGATACCGTTTCAGCTGCGTCCGGCATTTACCCTGCATTCGACACTGTCACATGTCAAACTGATCAGAAAA 
               
               
                   
                   
                 GGCGAGAGCGTCAGCTACGGAGCCGAGTACACAGCGGAAAAAGACACATGGATCGGGACGGTGCCTGTAGGCTATGCG 
               
               
                   
                   
                 GACGGCTGGCTCCGAAAATTGAAAGGGACCGACATCCTTGTGAAGGGAAAACGCCTGAAAATTGCCGGCCGAATTTGC 
               
               
                   
                   
                 ATGGACCAATTTATGGTGGAGCTGGATCAGGAATATCCGCCGGGCACAAAAGTCACATTAATAGGCCGGCAGGGGGAT 
               
               
                   
                   
                 GAATATATTTCCATGGATGAGATTGCAGGAAGGCTCGAAACCATTAACTATGAGGTGGCCTGTACAATAAGTTCCCGT 
               
               
                   
                   
                 GTTCCCCGTATGTTTTTGGAAAATGGGAGTATAATGGAAGTAAGAAATCCTTTATTGCAGGTAAATATAAGCAATTAA 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 18, 
                 GTGAAAAATAAATGGCTGTCTTTTTTTTCGGGTAAGGTCCAGCTTGAATTGACGGGAAGAGGGATTGAGCGGCTCCTT 
               
               
                   
                 yfqD gene 
                 AATGAATGCACAAGACAGGGGATTCCGGTCTTTCATGTCAAAAAAAAGAAAGAAGCCGTATCGTTATATATACAGCTT 
               
               
                   
                   
                 CAGGATGTACATGCCTTTCGGCGGGTAAGAAGTAAATTTAAATGTAAAGCCCGATTTATCAATCGGAAGGGATTTCCC 
               
               
                   
                   
                 TTCCTGTTGCTGAAATCAAAGCTGAATATAGGGTTTACGATCGGTTTTGCGATTTTTTTCATTCTTTTGTTTTTGCTG 
               
               
                   
                   
                 TCCAATATGGTGTGGAAAATTGATGTGACAGGCGCTAAGCCTGAAACAGAACATCAAATGAGGCAGCATCTTAATGAA 
               
               
                   
                   
                 ATCGGCGTCAAAAAGGGCCGTCTGCAGTTTTTAATGATGTCGCCCGAAAAAATACAGAAATCATTAACCAATGGAATA 
               
               
                   
                   
                 GACAATATCACTTGGGTCGGAGTTGATCTGAAGGGGACGACCATTCATATGAAAGTTGTGGAGAAAAATGAGCCCGAA 
               
               
                   
                   
                 AAAGAAAAATATGTTAGCCCGCGCAATATTGTCGCCAAAAAGAAAGCAACCATTACGAGAATGTTTGTGCAAAAAGGA 
               
               
                   
                   
                 CAGCCCATGGCCGCCATACACGATCATGTTGAAAAGGGACAGCTGCTTGTTTCGGGACTGATCGGCAGCGAAGACCAT 
               
               
                   
                   
                 CAGCAGGAAGTCGCCTCAAAAGCAGAAATTTATGGAGAAACCTGGTATAGATCAGAAGTGACAGTCCCGCTTGAAACA 
               
               
                   
                   
                 TTATTTAACGTCTATACGGGCAAAGTAAGGACAAAGCACAAGCTTTCTTTTGGTTCTTTGGCAATCCCGATCTGGGGG 
               
               
                   
                   
                 ATGACGTTTAAAAAAGAGGAATTGAAGCATCCAAAAACAGAACAAGAAAAGCATTCGCTTCATTTTCTCGGATTTAAG 
               
               
                   
                   
                 CTCCCTGTATCCTATGTCAAAGAGCAAACGAGAGAAAGTGAAGAGGCTTTGCGAAAATATACAAAAGAAGAAGCAGTT 
               
               
                   
                   
                 CAAGAAGGCATTAAATTGGGTAAACAGGATGTAGAGGATAAAATAGGCGAAAACGGCGAGGTGAAAAGTGAAAAAGTT 
               
               
                   
                   
                 TTGCACCAGACTGTTGAGAATGGTAAAGTAAAGTTGATTATTCTCTACCAAGTTATAGAAGATATCGTTCAAACCACA 
               
               
                   
                   
                 CCTATTGTCAGGGAGACTGAAGAATGA 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 19, 
                 TGACAATATGTCTCCTGTCATTATGTCCTTCACACTCTGATCAAACGTGACCAGCTGTTTTTCTTCCGTGAAATTCAT 
               
               
                   
                 EmR-comK cassette 
                 GACAAAAATATAATCATTGTCCTGATCCTGCCTCGCTTGTACGGAGACGCCTTTTCCGTGCCGAACCGGAAAAACTGG 
               
               
                   
                   
                 AGAGAGAGACAGGTCTGTGATCAGACCCTCATAGAAATCACGCTGAAATTGATCCTCCAAACGCGCGCCGATAAAATA 
               
               
                   
                   
                 CGCCTTGCCCTGCTGATACTCATGGCTTGTGACCGCTGGCGTGCGCGCATAAAAATCTTCTTGATACACCGCTTCCAC 
               
               
                   
                   
                 TGAAGCTGTCTTTACATCAATCACGGTTGCATAATCCTTCATTTCATATATTTGGCTGCGGTAGCTGACAGCGTTTCG 
               
               
                   
                   
                 ATCCTTCGGATACAGGGTGTCCGTTTCAAGAGGCTCAACTCCAAATATAGCTTGAAATCGATATCTCTGCAGTCGCGA 
               
               
                   
                   
                 TGATTAATTAATTCAGAACGCTCGGTTGCCGCCGGGCGTTTTTTATGCAGCAATGGCAAGAACGTCCCGGGGAGCTCC 
               
               
                   
                   
                 TAACTTATAGGGGTAACACTTAAAAAAGAATCAATAACGATAGAAACCGCTCCTAAAGCAGGTGCATTTTTTCCTAAC 
               
               
                   
                   
                 GAAGAAGGCAATAGTTCACATTTATTGTCTAAATGAGAATGGACTCTAGAAGAAACTTCGTTTTTAATCGTATTTAAA 
               
               
                   
                   
                 ACAATGGGATGAGATTCAATTATATGATTTCTCAAGATAACAGCTTCTATATCAAATGTATTAAGGATATTGGTTAAT 
               
               
                   
                   
                 CCAATTCCGATATAAAAGCCAAAGTTTTGAAGTGCATTTAACATTTCTACATCATTTTTATTTGCGCGTTCCACAATC 
               
               
                   
                   
                 TCTTTTCGAGAAATATTCTTTTCTTCTTTAGAGAGCGAAGCCAGTAACGCTTTTTCAGAAGCATATAATTCCCAACAG 
               
               
                   
                   
                 CCTCGATTTCCACAGCTGCATTTGGGTCCATTAAAATCTATCGTCATATGACCCATTTCCCCAGAAAAACCCTGAACA 
               
               
                   
                   
                 CCTTTATACAATTCGTTGTTAATAACAAGTCCAGTTCCAATTCCGATATTAATACTGATGTAAACGATGTTTTCATAG 
               
               
                   
                   
                 TTTTTTGTCATACCAAATACTTTTTCACCGTATGCTCCTGCATTAGCTTCATTTTCAACAAAAACCGGAACATTAAAC 
               
               
                   
                   
                 TCACTCTCAATTAAAAACTGCAAATCTTTGATATTCCAATTTAAGTTAGGCATGAAAATAATTTGCTGATGACGATCT 
               
               
                   
                   
                 ACAAGGCCTGGAACACAAATTCCTATTCCGACTAGACCATAAGGGGACTCAGGCATATGGGTTACAAAACCATGAATA 
               
               
                   
                   
                 AGTGCAAATAAAATCTCTTTTACTTCACTAGCGGAAGAACTAGACAAGTCAGAAGTCTTCTCGAGAATAATATTTCCT 
               
               
                   
                   
                 TCTAAGTCGGTTAGAATTCCGTTAAGATAGTCGACTCCTATATCAATACCAATCGAGTAGCCTGCATTCTTATTAAAA 
               
               
                   
                   
                 ACAAGCATTACAGGTCTTCTGCCGCCTCTAGATTGCCCTGCCCCAATTTCAAAAATAAAATCTTTTTCAAGCAGTGTA 
               
               
                   
                   
                 TTTACTTGAGAGGAGACAGTAGACTTGTTTAATCCTGTAATCTCAGAGAGAGTTGCCCTGGAGACAGGGGAGTTCTTC 
               
               
                   
                   
                 AAAATTTCATCTAATATTAATTTTTGATTCATTTTTTTTACTAAAGCTTGATCTGCAATTTGAATAATAACCACTCCT 
               
               
                   
                   
                 TTGTTTATCCACCGAACTAAGTTGGTGTTTTTTGAAGCTTGAATTAGATATTTAAAAGTATCATATCTAATATTATAA 
               
               
                   
                   
                 CTAAATTTTCTAAAAAAAACATTGAAATAAACATTTATTTTGTATATGATGAGATAAAGTTAGTTTATTGGATAAACA 
               
               
                   
                   
                 AACTAACTCAATTAAGATAGTTGATGGATAAACTTGTTCACTTAAATCAAAGGGGGAAATGACAAATGGTCCAAACTA 
               
               
                   
                   
                 GTGATATCTAAAAATCAAAGGGGGAAATGGGATCCAAAGGAGGCCATAATATGAGTCAGAAAACAGACGCACCTTTAG 
               
               
                   
                   
                 AATCGTATGAAGTGAACGGCGCAACAATTGCCGTGCTGCCAGAAGAAATAGACGGCAAAATCTGTTCCAAAATTATTG 
               
               
                   
                   
                 AAAAAGATTGCGTGTTTTATGTAAACATGAAGCCGCTGCAAATTGTCGACAGAAGCTGCCGATTTTTTGGATCAAGCT 
               
               
                   
                   
                 ATGCGGGAAGAAAAGCAGGAACTTATGAAGTGACAAAAATTTCACACAAGCCGCCGATCATGGTGGACCCTTCGAACC 
               
               
                   
                   
                 AAATCTTTTTATTCCCTACACTTTCTTCGACAAGACCCCAATGCGGCTGGATTTCCCATGTGCATGTAAAAGAATTCA 
               
               
                   
                   
                 AAGCGACTGAATTCGACGATACGGAAGTGACGTTTTCCAATGGGAAAACGATGGAGCTGCCGATCTCTTATAATTCGT 
               
               
                   
                   
                 TCGAGAACCAGGTATACCGAACAGCGTGGCTCAGAACCAAATTCCAAGACAGAATCGACCACCGCGTGCCGAAAAGAC 
               
               
                   
                   
                 AGGAATTTATGCTGTACCCGAAAGAAGAGCGGACGAAGATGATTTATGATTTTATTTTGCGTGAGCTCGGGGAACGGT 
               
               
                   
                   
                 ATTAGAAAAATAGCCGCGGGCGGCCGCACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCAT 
               
               
                   
                   
                 GAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACC 
               
               
                   
                   
                 TGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTTCAAGA 
               
               
                   
                   
                 ATTGATCCTCTAGCACAAAAAGAAAAACGAAATGATACACCAATCAGTGCAAAAAAAGATATAATGGGAGATAAGACG 
               
               
                   
                   
                 GTTCGTGTTCGTGCTGACTTGCACCATATCATAAAAATCGAAACAGCAAAGAATGGCGGAAACGTAAAAGAAGTTATG 
               
               
                   
                   
                 GAAATAAGACTTAGAAGCAAACTTAAGAGTGTGTTGATAGTGCAGTATCTTAAAATTTTGTATAATAGGAATTGAAGT 
               
               
                   
                   
                 TAAATTAGATGCTAAAAATTTGTAATTAAGAAGGAGTGATTACATGAACAAAAATATAAAATATTCTCAAAACTTTTT 
               
               
                   
                   
                 AACGAGTGAAAAAGTACTCAACCAAATAATAAAACAATTGAATTTAAAAGAAACCGATACCGTTTACGAAATTGGAAC 
               
               
                   
                   
                 AGGTAAAGGGCATTTAACGACGAAACTGGCTAAAATAAGTAAACAGGTAACGTCTATTGAATTAGACAGTCATCTATT 
               
               
                   
                   
                 CAACTTATCGTCAGAAAAATTAAAACTGAATACTCGTGTCACTTTAATTCACCAAGATATTCTACAGTTTCAATTCCC 
               
               
                   
                   
                 TAACAAACAGAGGTATAAAATTGTTGGGAGTATTCCTTACCATTTAAGCACACAAATTATTAAAAAAGTGGTTTTTGA 
               
               
                   
                   
                 AAGCCATGCGTCTGACATCTATCTGATTGTTGAAGAAGGATTCTACAAGCGTACCTTGGATATTCACCGAACACTAGG 
               
               
                   
                   
                 GTTGCTCTTGCACACTCAAGTCTCGATTCAGCAATTGCTTAAGCTGCCAGCGGAATGCTTTCATCCTAAACCAAAAGT 
               
               
                   
                   
                 AAACAGTGTCTTAATAAAACTTACCCGCCATACCACAGATGTTCCAGATAAATATTGGAAGCTATATACGTACTTTGT 
               
               
                   
                   
                 TTCAAAATGGGTCAATCGAGAATATCGTCAACTGTTTACTAAAAATCAGTTTCATCAAGCAATGAAACACGCCAAAGT 
               
               
                   
                   
                 AAACAATTTAAGTACCGTTACTTATGAGCAAGTATTGTCTATTTTTAATAGTTATCTATTATTTAACGGGAGGAAATA 
               
               
                   
                   
                 ATTCTATGAGTCGCTTTTGTAAATTTGGAAAGTTACACGTTACTAAAGGGAATGTAGATAAATTATTAGGTATACTAC 
               
               
                   
                   
                 TGACAGCTTCCAAGGAGCTAAAGAGGTCCCTAGACTCTAGACCCGGGGATCTCTGCAGTCGGGAAGATCTGGTAATGA 
               
               
                   
                   
                 CTCTCTAGCTTGAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCG 
               
               
                   
                   
                 GTGAACGCTCTCCTGAGTAGGACAAATCCGCCGCTCTAGCTAAGCAGAAGGCCATCCTGACGGATGGCCTTTTTGCGT 
               
               
                   
                   
                 TTCTACAAACTCTTGTTAACTCTAGAGCTGCCTGCCGCGTTTCGGTGATGAAGATCTTCCCGATGATTAATTAATTCA 
               
               
                   
                   
                 GAACGCTCGGTTGCCGCCGGGCGTTTTTTATGCAGCAATGGCAAGAACGTTGCTCTAGAGCGGCCGCATCGATTCACA 
               
               
                   
                   
                 GTGGCAATCTCCCCCGTATTCGTTTGAAATGTGCCACATTAACAGCGCCGGGTGATGTCCGTATCGTTCTGCTAATAA 
               
               
                   
                   
                 GCGGTTGATGTGCCGTGTTTTTTCTCGGTAGACTTTAGATGTGAGGCAGTGGTTGTGCCTTCCGCCGTGCAGCTGTTT 
               
               
                   
                   
                 GACGCGGGAGGCATTGACGCGCAAAACTTCCGGATAGGTTTGCGACAGCCAGGCCGGACGGGCTCCGCTCGGCGTTGC 
               
               
                   
                   
                 TAATATGACCCGGCCGCCTATACTGTGAATCCGCTCAAAAATATCATCCAGCCAT 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 20, 
                 5-TTACCTTCTCTCTTCTAAGTACCGTTCGTATAGCAT-3 - 
               
               
                   
                 primer P1 
                 lox7l-spc-lox66 cassette 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 21, 
                 5-CAAGCAAAGCTGTTTTATCTACCGTTCGTATAATGT-3 - 
               
               
                   
                 primer P2 
                 lox7l-spc-lox66 cassette 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 22, 
                 5-TACAAAGCAAAAGCGAAAATGACCATC-3 - 
               
               
                   
                 primer P3 
                 Upstream homology arm 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 23, 
                 5-ATGCTATACGAACGGTACTTAGAAGAGAGAAGGTAA-3 - 
               
               
                   
                 primer P4 
                 Upstream homology arm 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 24, 
                 5-ACATTATACGAACGGTAGATAAAACAGCTTTGCTTG-3 - 
               
               
                   
                 primer P5 
                 Downstream homology arm 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 25, 
                 5-CAGCTGATAGGATTCTTGCTCGCTTA-3 - 
               
               
                   
                 primer P6 
                 Downstream homology arm 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 26, 
                 5-TGATAGGTGGTATGTTTTCGCTT-3 - 
               
               
                   
                 primer P7 
                 Promoter p43 - 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 27 
                 5-ATAAATACCATGCTTCATGTGTACATTCCTCTCTTA-3 - 
               
               
                   
                 primer P8 
                 Promoter p43 - 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 28, 
                 5-TAAGAGAGGAATGTACACATGAAACATGGTATATAC-3 - 
               
               
                   
                 primer P9 
                 Primers DPEase Cc 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 29, 
                 5-GAATTCTTAGGAGTGTTTATGACATTC-3 - 
               
               
                   
                 primer P10 
                 Primers DPEase Cc 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 30, 
                 5-TAGAATGCAAAAAGTGAAATCATAATGATAGGTGGTATGTTTTCGCTTGA-3 - 
               
               
                   
                 primer P11 
                 P43-DPEase expression cassette 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 31, 
                 5-CGTCTGTACGTTCCTTAAGGAATTCTTAGGAGTGTTTATGACATTCTAAT-3 - 
               
               
                   
                 primer P12 
                 P43-DPEase expression cassette 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 32, 
                 5-ATTAGAATGTCATAAACACTCCTAAGAATTCCTTAAGGAACGTACAGACG-3 - 
               
               
                   
                 primer P13 
                 pUB110 vector backbone (according to P43-DPEase expression cassette) 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 33, 
                 5-TCAAGCGAAAACATACCACCTATCATTATGATTTCACTTTTTGCATT-3 - 
               
               
                   
                 primer P14 - 
                 pUB110 vector backbone (according to P43-DPEase expression cassette) 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 34, 
                 5-AAATCTAAAATTATCTGAAAAGGGAAGATCTTTCTAAAGAGGAAATGGTG-3 - 
               
               
                   
                 primer P15 - 
                 D-alanine racemase gene 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 35, 
                 5-TTGCTGAACAGATTAATAATAGATTGAATTCTCCATTTAGGTAAGTTAAT-3 - 
               
               
                   
                 primer P16 
                 D-alanine racemase gene 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 36, 
                 5-ATTAACTTACCTAAATGGAGAATTCAATCTATTATTAATCTGTTCAGCAA-3 - 
               
               
                   
                 primer P17 - 
                 PpuB110 vector backbone (according to D-alanine racemase) 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 37, 
                 5-CACCATTTCCTCTTTAGAAAGATCTTCCCTTTTCAGATAATTTTAGATTT-3 - 
               
               
                   
                 primer P18 
                 PpuB110 vector backbone (according to D-alanine racemase) 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 38 
                 AGCGGTACCATTATAGGT   C   ATG AAACATGGTATATACTACGCATATTGG 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 39 
                   AGCGGTACCATTATAGGT     ATG AAACATGGTATATACTACGCATATTGG 
               
               
                   
                   
               
               
                   
                 SEQ ID NO: 40 
                   AGCGGTACCATTATAGGT     ATG AAACATGGTATATACTACGCATATTGG 
               
               
                   
                   
               
            
           
         
       
     
    
    
     
       FIGURES 
         FIG.  1    represents an example of a strategy for the deletion of the alrA structural gene. 
         FIG.  2    represents the construction of the plasmid pUB-P43-DPEase-alrA also named vector/plasmid pR1. 
         FIG.  3    represents an outline of the vectors/plasmids pR1/pR2/pR3. The sequence region modified with respect to translational efficiency in pR2/pR3 is outlined as a black box. 
         FIG.  4    represents a PCR analysis of the beta-galactosidase genomic locus (ganA1/ganA2; wild type product: 2.1Kb). DNA was applied from three independent colonies of BsR, and two collection strain as  B. subtilis  1A751 and type 168 strain; M1, gene ruler 100 bp; M2, gene ruler 1 Kb ladder. 
         FIG.  5    represents a flow scheme for the cassette EmR-ComK removal using MazF cassette. X indicates on crossing-over event. 
         FIG.  6    represents a PCR analysis of the EmR-ComK cassette in BsR clones using gan locus specific primers. 1: BsR original strain, 2-5: Em sensitive clones, M: GeneRuler 1 kb ladder. 
         FIG.  7 A  represents a PCR analysis of D-alanine auxotrophic yqfD (BsR4) mutant candidate clones using specific yqfD region primers. 
         FIG.  7 B  represents a genetic setup of sporulation locus yqfD before and after the deletion and location of analytic primers. 1-5 BsR4. #1-5 (1.7 kb product indicates deletion of yqfD); 6: BsR original strain expected for yqfD wild type); M: GeneRuler 1 kb ladder. 
         FIG.  8    represents a phenotype analysis of ΔyqfD (BsR4) on LB+D-alanine supplementation.  FIG.  8 A  represents the BsR4 strain and  FIG.  8 B  represents the BsR strain. For each figure, the left side is before heat treatment, and the right side is after heat treatment. 
         FIG.  9    represents the phenotypic screening of BsR5 mutant candidates via loss of D-alanine prototrophy. Clones that have successfully excised the integrated mutagenesis cassette should no longer be able to grow on LB ( FIG.  9 B ) but strictly depend on medium supplemented with D-alanine ( FIG.  9 A ). 
         FIG.  10    represents a schematic overview of the strain platform filiation and genetic events applied. 
         FIG.  11    represents an overview of the Working Cell Bank preparation 
         FIG.  12    represents an overview of the strain cultivation providing the D-psicose 3-epimerase and its stabilization step. 
     
    
    
     The following Examples are provided in order to demonstrate and further illustrate certain embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof. 
     EXAMPLES 
     Example 1: Construction of a Recombinant  Bacillus subtilis  Producing a D-Psicose Epimerase from  Clostridium cellulolyticum  H10 
     Within a large part of the bacteria, D-alanine is an important component of the glycan subunits to form the cell wall (peptidoglycan). 
     Alanine is usually found as the L-stereoisomer in nature, making the conversion to D-alanine by the cytoplasmic D-alanine racemase (alrA) essential for cell growth. 
     Lack of the enzyme leads to rapid cell lysis due to a failure in the initial step of peptidoglycan biosynthesis. 
     The entire alrA structural gene (GenBank, no. CAB12271.1) and regulatory signals for its expression were contained within the 1.17 kb DNA fragment (SEQ ID NO: 17). 
     1. Construction of the  Bacillus subtilis  Host Named BsR 
     Fusion of the antibiotic resistance marker cassette with long-flanking homology regions by PCR was done as described by Shevchuk et al. (Nikolai A. Shevchuk et al. Nucleic Acids Research, 2004(32): e19). In brief, it was carried out as follows. 
     The lox71-spc-lox66 cassette was amplified from vector p7S6 using P1/P2 primer pair. Two additional primer pairs (P3/P4 and P5/P6) were used to amplify about 900 bp DNA fragments flanking the D-alanine racemase region for deletion at its front and back ends. 
     Extensions of 32 nucleotides (nt) that were complementary to the 5′ and 3′ ends of the amplified marker cassette were added to the 5′ end of the reverse and forward primers of the front and back flanking regions, respectively. Finally, the two flanking homology regions and the lox71-spc-lox66 cassette were fused by PCR. 
     The PCR product was directly transformed into the  B. subtilis  host (the PCR product has been recombined with the  B. subtilis  chromosome due to the two flanking homology fragments). 
     Transformants clones were selected on LB agar enriched with both spectinomycin (Spc) (100 μg/mL) and D-alanine (200 μg/mL). 
     A positive clone which provides the phenotype [alrA − ; Spc R ] was selected for further modification. 
     Then the antibiotic-resistant gene Spc was knocked out by the Cre/Lox system. 
     Finally, a  Bacillus subtilis  host [alrA − ] in which the alanine racemase alrA gene is deleted is obtained ( FIG.  1   ). This  Bacillus subtilis  is named BsR. 
     2. Construction of the Recombinant Plasmid and the Antibiotic Free  B. subtilis  DPEase Producer 
     The  Bacillus subtilis  endogenous promotor P43 was amplified from the well-known strain  Bacillus subtilis  168 chromosome using the primer pair P7/P8. The D-psicose 3-epimerase gene of  Clostridium cellulolyticum  H10 (ATCC 35319) (GenBank no CP001348.1) (sequence II) encoding the protein with locus tag YP_002505284 was de novo synthetized by with 1) integration of NdeI and XhoI restriction site at 5′ and 3′terminus (for further gene cloning steps) and 2) a nucleotide substitution T558C to neutralize a NdeI restriction site (SEQ ID NO: 4). 
     The P43 promoter and D-psicose 3-epimerase gene were fused as an expression cassette via SOE-PCR (splicing overlap extension PCR) using P7 and P10 primers. Then the PCR-produced p43-DPEase cassette was cloned into pMD-19T vector. 
     The pUB110 plasmid was used with its original HpaII promotor in order to improve the expression. 
     The plasmid antibiotic resistance gene-free was constructed referring a method called simple cloning (Chun You et al. Appl. Environ. Microbiol. 2012, 78(5): 1593-1595) which is a sequence-independent method without the need for restriction and ligation enzymes. 
     The protocol consists of three steps: 
     (1) Linear DNA (P43-DPEase expression cassette and the appropriate zone of linear pUB110 vector backbone (the fragment outside Mob gene region)) were separately amplified by PCR with primers P11/P12 and P13/P14 respectively (P11/P12 contain the 40-50 bp overlapping termini of P13/P14). 
     (2) The DNA multimers was generated based on these DNA templates (target gene and corresponding vector) by POE-PCR (prolonged overlap extension PCR) without primers and 
     (3) the POE-PCR products (pUB-P43-DPEase) were transformed into the  Bacillus subtilis  competent cells. Hit transformants were recovered on LB agar by adding 50 μg/mL kanamycin. Using the same method, D-alanine racemase gene was inserted replacing the Kanamycin (Km) and Bleomycin (Blm) antibiotic-resistant genes region. 
     D-alanine racemase gene and vector backbone were amplified via PCR with the P15/P16 and P17/P18 primers respectively. 
     The DNA multimers were transformed within  Bacillus subtilis  [alrA − ] competent cells, deficient in biosynthesizing D-alanine metabolite. 
     Finally, the plasmid pUB-P43-DPEase-alrA (SEQ ID NO: 14) ( FIG.  2   ) was selected on LB agar without adding D-alanine. 
     The main advantage of this strategy is to provide direct selection for the plasmid in complex media without antibiotics. 
     As the D-alanine racemase involved in the cell wall metabolism, the loss of the activity leads to the cell lysis, preventing the accumulation of a population of cells which have lost the plasmid. 
     Example 2: Plasmid Optimization for Higher DPEase Expression 
     The experimental strategy has aimed at revealing the expression potential and intrinsic limitations of  Bacillus subtilis  as DPEase expression host (BsR), as obtained above. 
     The modifications introduced into the parental plasmid pUB-P43-DPEase-alrA (pR1) target by a translational efficiency (pR2, pR3). 
     This means for pR2/pR3, if the gene expression is “on” in a given cell at a given time point, more protein should be expected to be delivered at this moment. 
     1. Plasmid Optimization for the Ribosome Binding Sites (pR2) 
     As a template for generation of optimized DPEase expression constructs, the plasmid pUB-P43-DPEase-alrA (or pR1) was isolated from overnight cultivation in standard LB medium and the plasmid free strain was kept for further steps. 
     These plasmid preparations served as templates for PCR mediated insertion of variant ribosome binding sites and adjacent regions ( FIG.  3   ). After successful mutagenesis PCR, the new plasmid was introduced back to the  B. subtilis  alrA deficient plasmid-free strain (BsR). 
     Successfully transformed clones were cultivated in standard LB medium and pass throughout a primary activity screening phase (Protocol #1). 
     Then, a plasmid DNA was prepared from overnight cultivations for electrophoresis and sequencing verification of the ribosome binding site zone change. 
     The upstream sequence identified in the pR2 clone that performs best in conjunction with the downstream DPEase open reading frame is shown below. 
     Nucleotide sequence of the 5′ untranslated region upstream of the DPEase in pR1 (1) and pR2 (2). The ATG codon of the DPEase gene is shown underlined and the RBS modified region is in italic bold in Table 1 below. 
     
       
         
           
               
             
               
                 TABLE 1 
               
               
                   
               
               
                 Nucleotide sequences of the 5′ untranslated 
               
               
                 region upstream of the DPEase in pR1 (1) 
               
               
                 and pR2 (2) 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                   
                 pR1 
                 1- AGCGGTACCATTATAGGT   ATG   
               
               
                   
                   
                 AAACATGGTATATACTACGCATATTGG (SEQ ID NO: 
               
               
                   
                   
                 38) 
               
               
                   
                   
               
               
                   
                 pR2 
                 2- AGCGGTACCATTATAGGT   ATG   
               
               
                   
                   
                 AAACATGGTATATACTACGCATATTGG (SEQ ID NO: 
               
               
                   
                   
                 39) 
               
               
                   
                   
               
            
           
         
       
     
     Plasmid pR2 of SEQ ID NO: 15 contains an optimized sequence of SEQ ID NO: 1 or SEQ ID NO: 39. 
     Protocol #1: Enzymatic Detection of DPEase Activity 
     The analysis of DPEase screening samples was performed by applying a Fructose/Glucose Assay Kit from Megazymes (K-FRUGL). 
     Initial evaluation revealed that psicose does not give rise to any signal, thus, DPEase activities can be measured by following the reduction of fructose contents in the reactions. Briefly, samples were diluted 1:1000 freshly prior to the reaction. 
     Calibration glucose/fructose standards as well as a fructose/PBS mix were always included. Sugars could be detected in a linear range of 0-100 mg/L. 
     100 μL sample were transferred to an assay-plate (96 well MTP, flat-bottom). 90 μL reaction mix 1+2 (10 μL each of Solution 1&amp;2, +70 μL milliQ (mQ) water) was added and allowed to incubate at RT for a few minutes. 
     20 μL reaction mix 3 (2 μL Solution 3+18 μL mQ water) was added and after 5 min the OD340 was read out as “blank” 20 μL reaction mix 4 (2 μL Solution 4+18 μL mQ water) was added and after 5 min the OD340 was read out as residual fructose. 
     The residual fructose was calculated with the help of the calibration standards, and the converted psicose estimated in comparison to the untreated fructose sample. 
     2. Establishment of Vector with Customized Translation Initiation (pR3) 
     The previous pR2 variant depicted in  FIG.  3    served as parental plasmid for further optimization of the translation initiation region (spacer). 
     To this end, the proximal 4 nucleotides upstream of the DPEase open reading frame were randomized via PCR mutagenesis. 
     The resulting plasmids variants were introduced back to the  B. subtilis  alrA deficient plasmid-free strain (BsR) and cultivated onto standard LB agar plates. 
     In order to cover all possible 4 nucleotide combinations, a mutant bank of above 2000 clones was randomly picked and cultivated in 96-Deep well plates (DWP and assessed for DPEase expression in the primary activity screening phase (Protocol #1). 
     The best clone harboring the pR3 plasmid has been sequenced. (below) 
     Nucleotide sequences of the 5′ untranslated region upstream of the DPEase in pR1 (1) and pR2 (2) and pR3 (3) are shown in Table 2 below. The ATG codon of the DPEase gene is shown underlined and the RBS modified region is in italic bold and the translation initiation region boxed. 
     
       
         
           
               
             
               
                 TABLE 2 
               
               
                   
               
               
                 Nucleotide sequences of the 5′ untranslated 
               
               
                 region upstream of the DPEase in pR1 (1) 
               
               
                 and pR2 (2) 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
            
               
                 pR1 
                 1- AGCGGTACCATTATAGGT     ATG   
               
               
                   
                 AAACATGGTATATACTACGCATATTGG (SEQ ID NO: 
               
               
                   
                 38) 
               
               
                   
               
               
                 pR2 
                 2-  AGCGGTACCATTATAGGT     ATG   
               
               
                   
                 GAAACATGTATATACTACGCATATTGG (SEQ ID NO: 
               
               
                   
                 39) 
               
               
                   
               
               
                 pR3 
                 3-  AGCGGTACCATTATAGG     ATG   
               
               
                   
                 AAACATGGTATATACTACGCATATTGG (SEQ ID NO: 
               
               
                   
                 40) 
               
               
                   
               
            
           
         
       
     
     Plasmid pR3 of SEQ ID NO: 16 contains an optimized sequence of SEQ ID NO: 2 or SEQ ID NO: 40. 
     3. Expression Screening and Enzyme Assay 
     A second activity screening phase has been done for more representative DPEase production. For the re-assessment, a selection of best performing clones was chosen for cultivation with larger volume. 
     Thus, the strain BsR strain previously transformed with pR1 and pR2 and pR3 plasmids were cultivated in shake flasks (Table 3). 
     Samples were taken at final point (16 h) and cells were collected by centrifugation at 6000 g for 15 minutes and the supernatant was discarded. 
     The cells pellets harboring  C. cellulolyticum  DPEase prepared by freeze-drying were vacuum freeze-dried, grinded and directly used as an enzyme powder. 
     Next, DPEase activity for each enzyme powders produced was done (following the method given below). 
                     TABLE 3                  Media composition used for the DPEase production from plating       to production cultivations in shakeflasks at 37° C. at 200 rpm.                                         1 st  Seed   2 nd  Seed           Media comp.(g/L)   Plate   culture   culture   Production               Trypton from milk casein   10   10   10           (Biokar)                       Yeast Extract    5    5    5   15         (BactoYE Difco, BD)                       NaCl [7647-14-5]   10   10   10   8         Dextrose (Roquette Freres)               15         Na 2 HPO 4 , 12H 2 O                1         [10039-32-4]                       MgSO 4 , 7H 2 O                1         [10034-99-8]                       MnSO 4 , H 2 O [10034-96-5]                0.008       Antifoam (EROL18)               0.3       pH adjustment       no   no   no       (NaOH 4M) 7.4*               *pH is adjusted before heat sterilization. The effective cultivation initial pH is roughly 6.75            
Incubation time were overnight for the plate, 16 h for the first seed culture, up to Abs 600nm  for second seed culture and 16 h for the production.
 
     Method: DPEase Enzyme Assay Description 
     The DPEase activity was measured via determining the quantity of D-psicose produced using a whole-cell reaction. 
     One milliliter of the reaction mixture contained D-fructose (80 g/L) in 50 mM Tris-HCl, pH7.5, and 200 μL of enzyme solution; the cells were dissolved in tris-HCL. 
     The reaction was incubated at 60° C. for exactly 10 minutes and ended by boiling at 100° C. for exactly 10 minutes. The generated D-psicose in the mixture was detected via a Waters Alliance HPLC, fitted with aminex HPX-87Ca 2+  column (from Biorad) with dimensions 250×4 mm, #125-0094 and a refractive index detector (waters 410). 
     The column was eluted with pure water at a flow rate of 0.3 ml/min at 85° C. One unit of DPEase activity was defined as the amount of enzyme that catalyzed the production of 1 μmol of D-psicose per minute. 
     4. DPEase Performance Results 
     The best DPEase enzyme performances are gathered into the following Table 
     
       
         
           
               
             
               
                 TABLE 4 
               
             
            
               
                   
               
               
                 Results of strain BsR transformed with the plasmid pR1, pR2 or pR3 
               
            
           
           
               
               
               
            
               
                   
                 DPEase enzyme act. 
                   
               
               
                   
                 (U/mL) 
                 n 
               
               
                   
               
            
           
           
               
               
               
            
               
                 BsR-pR1 
                 10.57 
                 5 
               
               
                 BsR-pR2 
                 26.85 
                 10 
               
               
                 BsR-pR3 
                 38.85 
                 20 
               
               
                   
               
               
                 n means the number of assays performed. 
               
            
           
         
       
     
     Initial strain (BsR), which is D-alanine racemase deficient, harboring the constructed pUB-P43-DPEase-alrA vector (pR1) showed a DPEase enzyme activity of about 10.57. 
     The two steps plasmid optimizations showed higher DPEase activity with about 26.85 U/mL and 38.85 U/mL for RBS region change (pR2) and translation initiation spacer optimization (pR3), respectively. Plasmid pR3 is the most promising plasmid. 
     Example 3:  Bacillus subtilis  BsR Improvement for DPEase Enzyme Expression Enhancement 
     In parallel to the plasmid optimization, the strain itself, BsR, was optimized, especially for the regulatory and safety purposes. 
     Antibiotics sensitivity of the BsR showed the strain was able to grow when erythromycin was added at 5 μg/mL. This observation clearly indicates that the strain was erythromycin resistant (Em R ). This resistance has to be removed.  Bacillus  genus bacteria are known to produce a dedicated, very resistant and non-reproductive structure to enter in a state of dormancy: the endospores. 
     Bacterial endospores keeps all material the cell needs to recover a living cell when favorable conditions will appear. 
     The endospores are the perfect dissemination factor for the strain and is a serious risk for environmental and health contamination. For industrial uses of an endospore forming BsR, it is important to abort the endospore forming pathway. 
     1. Removal of the EmR-comK Cassette: Generation of BsR3 
     Aiming to develop an enzyme producer strain by molecular biology tools, the  Bacillus subtilis  BsR was tested for the applicability of different antibiotics (tetracycline, erythromycin and kanamycin) and sugars (xylose and mannitol) likely used as inducers of gene expression on some plasmids. 
     Surprisingly, BsR was able to cultivate on erythromycin even at a concentration that is applied for high copy plasmids (5 μg/mL) selection pressure and the strain showed a clear delayed cultivation on xylose, compare to  Bacillus subtilis  (wild-type). 
     As the  B. subtilis  beta-galactosidase gene lacA (also named ganA) can serve as integration site for heterologous expression cassettes and/or as a reporter gene to test promotor induction efficiencies, its functionality was tested on X-gal agar plate. 
     X-gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside(C 14 H 15 BrClNO 6 )) which is an analog of lactose sensitives to beta-galactosidase (the enzyme cleaves the beta-glycosidic bond in D-lactose) is cleaved and galactose and 5-bromo-4-chloro-3-hydroxyindole are released. 
     The latter spontaneously dimerizes and is oxidized into 5,5′-dibromo-4,4′-dichloro-indigo (insoluble blue color). 
     Indeed, native lacA gene by growing the cells on agar containing the chromogenic substrate X-gal should have blue colored colonies, indicating the lacA gene is active. For BsR strain, no blue colonies were seen onto X-gal plate. 
     Thus, lacA PCR analysis was done compared to a  B. subtilis  strains (wild-type). 
     If wild type lacA gene is present, a 2.1 kb product should be provided. PCR analysis clearly showed a larger amplification band of about 5 kb indicating the lacA locus contained an insert in ( FIG.  4   ). 
     This amplified fragment was amplified and blasted to reveal the existence of a cassette containing the EmR gene and a comK gene controlled by the xylose-inducible promoter PxylA. 
     To remove the EmR-comK cassette (PCR fragment of 6.2 kb), an  Escherichia coli  toxin gene MazF as a counter-selectable marker was used. 
     The MazF gene was placed under the control of an isopropyl-O-d-thiogalactopyranoside (IPTG)-inducible expression system and associated with the alrA gene to form the MazF cassette, which was flanked by three targeting sequences. 
     A double-crossover event between delivery vector and the chromosome integrated the MazF cassette in front of the targeted EmR-comK cassette, and yielded an IPTG-sensitive strain with D-alanine racemase. Another single-crossover event between the two ganA sequences led to the excision of the MazF cassette ( FIG.  5   ). 
     Then clones were evaluated regarding the desired phenotypes of successful mutants a) no growth with erythromycin selection and b) no growth on medium lacking D-alanine. 
     The latter clones were successfully checked via PCR analysis for the desired EmR-comK cassette removal genotype with a 2.3 kB amplified fragment ( FIG.  6   ). 
     Theses erythromycin sensitive (Em S ) and D-alanine auxotrophic clones were subsequently transformed with the DPEase expression plasmid pR3. 
     The resulting clones were able to growth on LB with no external D-alanine supplementation. 
     2. Spore Inactivation: Generation of BsR4 and BsR5 
     Previously to generate the BsR5 strain version which is erythromycin sensitive and sporulation deficient (double mutant Em S  Spo − ), the impact of the endospore inactivation was evaluated with the strain BsR (containing EmR-comK cassette) leading to the single mutant named BsR4, Em R  spo −  genotyped. 
     The strategy to disrupt the sporulation metabolic cascade was to delete the yqfD essential gene, which acts during the stage IV (one of the later phase on sporulation process) of the endospore maturation, in order to abort the sporulation. 
     a—Generation of the Single Mutant Strain, BsR4 
     Establishment of a D-alanine racemase selectable mutagenesis cassette for deletion of the sporulation gene yqfD was generated and introduced into BsR devoid of the DPEase harbored plasmid. 
     The alrA cassette was done as the one used for the EmR-ComK cassette removal, with specific sequence for ydfD gene deletion. 
     Transformants were successfully selected by their capability to grow on medium with no D-alanine in. 
     These candidates were applied for IPTG induced counter selection that leads to clones devoid of the mutagenesis cassette as well as the yqfD sporulation gene (ΔyqfD). 
     The single mutants were identified by their D-alanine auxotrophy and by PCR analysis of the yqfD locus ( FIG.  7   ). 
     In order to evaluate the sporulation phenotype of BsR4 strain, the mutant clones were cultivated in LB+D-alanine medium for overnight growth. 
     Cultures were then spotted on sporulation agar plates (supplemented with D-alanine) to form large colonies. 
     The sporulation plates were incubated at 37° C. for 3 days and evaluated by microscopy. The BsR original strain had produced phase-bright spores, while the ΔyqfD mutant clones did not produce any phase bright spores indicating the sporulation defect (spores produced by mutants were dark instead of bright which indicates that they are unable to proceed to maturation). 
     To check that the mutant clones were not able to produce any mature (so viable) endospores, an overnight cultivation in LB+D-alanine was performed at 37° C. The day after, 2×0.5 mL were sterile sampled into sterile tubes. 
     The first tube was directly spotted on a LB+D-alanine medium when the second was incubated at 80° C. for 30 minutes. 
     Heat treatment aims to kill vegetative cells, and only mature endospores can survive. 
     After the heat treatment, the broth was spotted onto the previous described plate (directly next to the previous unheattreated spots). 
     The plate was then incubated overnight at 37° C. for growth. As expected, only BsR wild type clone survived the heat treatment. 
     Only cellular debris was visible for the spots after heat treatment for BsR4 clone ( FIG.  8   ). 
     b—Generation of the Double Mutant Strain BsR5 
     The mutagenesis cassette targeting the sporulation locus yqfD that has already been successfully applied to generate the single mutant strain, BsR4, was introduced into the erythromycin sensitive strain, BsR3. 
     After successful genomic integration, mutant screening was initiated for the identification of clones that had excised the mutagenesis cassette from the genome, leading to clean deletion of yqfD gene. 
     As performed for BsR4 strain, the clones were selected for their inability to produce mature endospores. After an overnight cultivation, samples were spotted before and after the heat treatment onto LB+D-alanine plates then incubated for another night at 37° C. 
     The hit candidates that did not grow after heat treatment were picked and spotted to LB medium plate for their loss of D-alanine prototrophy and incubate overnight at 37° C. 
     The hits candidates were those which showed growth ( FIG.  9   ). 
     Finally, an industrial strain platform, BsR5, was obtained as a double mutant erythromycin sensitive and sporulation negative for respect environmental and safety regulations ( FIG.  10   ) 
     3. DPEase Enzyme Production Performance Results 
     All the strains obtained (BsR3, BsR4 and BsR5) were transformed with hit plasmid pR3. They were cultivated regarding the following protocol ( FIGS.  11  and  12   ): 
     Working Cell Bank Construction: 
     Working cell bank refers to a −80° C. frozen stock, in Nalgene® vials of 2 mL. 
     The process contains a petri dish cultivation on LB medium (trypton 10 g/L, Yeast extract 5 g/L, NaCl 5 g/L, pH 7.5 adjusted with 10 N soda) at 37° C. for 16 h. A cellular suspension is prepared within a 5 or 10 mL of liquid LB+0.1 mM manganese (MnCl 2 , 4H 2 O [13446-34-9]) medium to obtain a 10 O.D. 600nm  preparation. A 500 mL shake flask with 2 lateral baffles containing 50 mL liquid LB+0.1 mM manganese is sterilized at 121° C. for 21 minutes. The latter medium is inoculated to 0.1 O.D. 600nm  with the freshly interim suspension. The cultivation is incubated at 37° C. and 250 rpm (orbital=5 cm) and the growth is monitored with hourly O.D. 600nm  measurements. The procedure move one step ahead when the cultivation reaches O.D. 600nm  MAX/2. Then, the exact volume of the final culture is measured and the same volume of cryoprotectant (30% v/v) Glycerol [56-81-5]) is slowly added and mixed until good homogenization. The latter suspension is then aliquoted at 1.8 mL into 2 mL vials. The vials freshly filled up are rapidly stored into a −80° C. freezer and designed as a Working Cell Bank for further uses. 
     Strain Cultivation for DPEase Enzyme Production 
     As a seed culture, a 300 mL shake flask unbaffled was filled up with 30 mL LB medium supplemented with manganese and then heat sterilized at 121° C. for 20 minutes. 1.8 mL of a working cell bank tube was used for inoculation. The cultivation was incubated 4 h at 37° C. and 250 rpm (orbital=5 cm). 
     As a production cultivation, a 0.9 mL of the previous seed culture was used to inoculate a sterile 300 mL shake flask with 3 lateral baffles and 50 mL modified LB-ROQ medium (Dextrose monohydrate 15 g/L, Yeast extract 15/L, NaCl [7647-14-5] 8 g/L, K 2 HPO 4  [7758-11-4] 7 g/L, KH 2 PO 4  [7778-77-0] 1.3 g/L, MgSO 4 . 7H 2 O [10034-99-8] 50 mg/L, MnSO 4 . H 2 O [10034-96-5] 0.4 mg/L and MnCl 2 . 4H 2 O [13446-34-9] 19 mg/L. pH should be close to neutral. The culture was incubated at 37° C. and 250 rpm (orbital 5 cm) for 16 h. The DPEase enzyme assessment was done as detailed into example 2. 
     The best DPEase enzyme performances are gathered into the Table 5 below indicating the average value of the performance and the number of trials performed: 
     
       
         
           
               
             
               
                 TABLE 5 
               
             
            
               
                   
               
               
                 Results of the strain BsR3 transformed with the plasmid pR3,  
               
               
                 the strain BsR4 transformed with the plasmid pR3  
               
               
                 or the strain BsR5 transformed with the plasmid pR3  
               
               
                 n means the number of assays performed. 
               
            
           
           
               
               
               
               
            
               
                   
                   
                 Average value DPEase enzyme 
                   
               
               
                   
                   
                 act. (U/mL) 
                 n 
               
               
                   
                   
               
            
           
           
               
               
               
               
            
               
                   
                 BsR3-pR3 
                 39.25 
                 2 
               
               
                   
                 BsR4-pR3 
                 44.31 
                 2 
               
               
                   
                 BsR5-pR3 
                 52.06 
                 11 
               
               
                   
                   
               
            
           
         
       
     
     The successive DPEase enzyme productions with the different constructed strain platforms, BsR3 (single mutant Em S ), BsR4 (single mutant ΔyqfD) and BsR5 (double mutant Em S , ΔyqfD) when transformed with the plasmid pR3 (puB-P43-DPEase-alrA vector) leaded to progressively improved the performance. 
     Intermediate single mutation strains (BsR3 and BsR4) were assessed for the DPEase production to follow the impact of the genetic modifications. For these two strains, the performance was not affected. 
     The final strain, BsR5 transformed with the plasmid pR3, which is environmentally and safety optimized, leads to the better expression of the enzyme DPEase. 
     The strain might save resources expressing DPEase instead of produces erythromycin resistance tools and endospore full maturation processing machinery. 
     Example 4: Optimization of the Fermentation Medium for DPEase Enzyme Expression Enhancement 
     Material &amp; Methods 
     The strain used in the strain BsR5 transformed with the plasmid pR3. 
     1.1 Production of Biomass 
     The production of biomass begins with a preculture step. Glucose (15 g/L), yeast extract (15 g/L) and NaCl (15 g/L) are dissolved in demineralized water (QS 1 L). pH is not adjusted. The medium is placed in a baffled Erlenmeyer (2000 mL), then the erlenmeyers are autoclaved 20 minutes at 121° C., then inoculated in sterile conditions with 1 cryotube, then incubated at 37° C., during 4 hours, at 110 RPM. 
     The precultures are carried out in 2 L erlenmeyers containing 0.5 L of medium. The erlenmeyers are incubated for 3 h at 37° C. and 110 RPM so as to obtain an optical density of between 0.5 and 1 or a DCW (dry cell weight) of between 0.07 and 0.18 g/L. 
     The production step consists of a “batch” type fermentation which is carried out with a complex medium based on glucose, yeast extract and salts. The management of the pO2 is special since the medium is micro-aerated: the OUR (oxygen consumption) is maintained around 7 mmol/l/h. To do this, the agitation and the aeration are weak and fixed (200 RPM and 9 L/min), which causes a zero pO2 during the ¾ of the production. During the fermentation, there is no addition of medium (fed). A regulation of pH 6 is set up with ammonia 20% (w/w). 
     1.2 Biomass Preparation—Grinding 
     Biomass is collected when glucose is completely consumed. At this point the enzymatic activity is maximal. The biomass is then centrifuged (10000 g/5 min) and washed with a 50 mM PBS buffer pH8. The cells are then broken in a ball mill (30 min/2 g beads/1 g washed must). The mixture obtained is filtered through a 0.45 μm filter in order to remove the debris. The solution obtained is stable for 7 days at 4° C. 
     1.3 Measurement of Activity 
     Enzymatic analysis is carried out under the following conditions: 800 pi of substrate (fructose 400 g/L in 50 mM PBS pH 8) are preincubated at 55° C. for 5 minutes. The necessary amount of enzymatic solution is added to start the reaction. The whole is incubated for 10 min at 55°. The reaction is then stopped by a passage during 10 minutes at 100° C. The measurement of the psicose produced is carried out by HPLC (Ca2+ column at 65° C., H 2 O at 0.3 ml/min and refractometric detection) by measurement of the % area of psicose. The activity is expressed in μmol of psicose formed per ml of enzyme and per minute of reaction (U/ml). 
     Several fermentation medium were tested, and their compositions are detailed in Table 6 below. 
     
       
         
           
               
             
               
                 TABLE 6 
               
             
            
               
                   
               
               
                 Fermentation medium tested 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                   
                   
                   
                   
                 Time until 
                   
                   
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                 complete 
                 Oxygen 
               
               
                   
                   
                   
                   
                   
                   
                   
                 OUR 
                 glucose 
                 partial 
                 DPEase 
               
               
                   
                 Glucose 
                 Yeast 
                 (NH 4 ) 2 SO 4   
                 KH 2 PO 4   
                 MgSO 4   
                 MnSO 4   
                 maximal 
                 consumption 
                 pressure 
                 activity 
               
               
                 Reference 
                 (g/L) 
                 (g/L) 
                 (g/L) 
                 (g/L) 
                 (g/L) 
                 (mg/L) 
                 (mmol/h/L) 
                 (h) 
                 (PO 2 ) 
                 (U/mL) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
            
               
                 F2 160808 
                 15 
                 15 
                 1 
                 1 
                 1 
                 8 
                 8 
                 8 
                 No 
                 34.0 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 regulation 
               
               
                 F1 160811 
                 15 
                 15 
                 1 
                 1 
                 1 
                 8 
                 8 
                 9 
                 No 
                 40.0 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 regulation 
               
               
                 F2 160811 
                 15 
                 15 
                 1 
                 1 
                 1 
                 8 
                 8 
                 9 
                 No 
                 41.9 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 regulation 
               
               
                 F1 160817 
                 30 
                 30 
                 2 
                 2 
                 2 
                 16 
                 7 
                 16 
                 No 
                 41.8 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 regulation 
               
               
                 F2 160817 
                 30 
                 15 
                 1 
                 1 
                 1 
                 8 
                 7 
                 16 
                 No 
                 58.8 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 regulation 
               
               
                 F1 160823 
                 15 
                 15 
                 1 
                 1 
                 1 
                 8 
                 3 
                 16 
                 No 
                 28.2 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 regulation 
               
               
                 F2 160823 
                 15 
                 15 
                 1 
                 1 
                 1 
                 8 
                 3 
                 13 
                 No 
                 14.2 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 regulation 
               
               
                 F1 160906 
                 45 
                 15 
                 1 
                 1 
                 1 
                 8 
                 8 
                 23 
                 No 
                 91.9 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 regulation 
               
               
                 F2 160906 
                 30 
                 15 
                 1 
                 1 
                 1 
                 8 
                 8 
                 17 
                 No 
                 71.8 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 regulation 
               
               
                 F1 160919 
                 Fed 
                 15 
                 1 
                 1 
                 1 
                 8 
                 8 
                 23 
                 No 
                 121.2 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 regulation 
               
               
                 F2 160919 
                 60 
                 15 
                 1 
                 1 
                 1 
                 8 
                 8 
                 28 
                 No 
                 139.9 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 regulation 
               
               
                 F1 160926 
                 Fed 
                 15 
                 1 
                 1 
                 1 
                 8 
                 8 
                 27 
                 No 
                 143.4 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 regulation 
               
               
                 F2 160926 
                 45 
                 15 
                 1 
                 1 
                 1 
                 8 
                 8 
                 22 
                 No 
                 128.0 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 regulation 
               
               
                 F1 161003 
                 45 
                 15 
                 1 
                   
                 1 
                 8 
                 8 
                 20 
                 No 
                 127.7 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 regulation 
               
               
                 F2 161005 
                 45 
                 15 
                 1 
                 1 
                 1 
                 8 
                 8 
                 21 
                 No 
                 134.1 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 regulation 
               
               
                 F1 161011 
                 45 
                 15 
                 1 
                 1 
                 1 
                 8 
                 9 
                 21 
                 No 
                 133.8 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 regulation 
               
               
                 F2 161011 
                 100  
                 15 
                 1 
                 1 
                 1 
                 8 
                 8 
                 71 
                 No 
                 156.6 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 regulation 
               
               
                 F1 161026 
                 60 
                 15 
                 1 
                 1 
                 1 
                 8 
                 80 
                 15 
                 Regulated 
                 71.7 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 5% 
               
               
                 F2 161026 
                 60 
                 15 
                 1 
                 1 
                 1 
                 8 
                 20 
                 17 
                 No 
                 134.7 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 regulation 
               
               
                 F1 161107 
                 Fed 
                 15 
                 1 
                 1 
                 1 
                 8 
                 8 
                 32 
                 No 
                 143.0 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 regulation 
               
               
                 F2 161107 
                 60 
                 15 
                 1 
                 1 
                 1 
                 8 
                 7 
                 32 
                 No 
                 133.5 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 regulation 
               
               
                 F1 161122 
                 Fed 
                 15 
                 1 
                 1 
                 1 
                 8 
                 25 
                 29 
                 No 
                 166.7 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 regulation 
               
               
                 F2 161122 
                 60 
                 15 
                 1 
                 1 
                 1 
                 8 
                 3 
                 60 
                 No 
                 129.3 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 regulation 
               
               
                 F2 170117 
                 Fed 
                 15 
                 1 
                 1 
                 1 
                 8 
                 15 
                 35 
                 No 
                 125.6 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 regulation 
               
               
                 F1 170124 
                 Fed 
                 15 
                 1 
                 1 
                 1 
                 8 
                 60 
                 24 
                 Regulated 
                 41.2 
               
               
                   
                   
                   
                   
                   
                   
                   
                   
                   
                 5% 
               
               
                   
               
            
           
         
       
     
     Thus, a fermentation medium comprising 60 g/L (medium called “F2 160919”) leads to a DPEase activity of about 139.9 U/mL whereas a fermentation medium comprising 15 g/L (medium called “F1 160811”) leads to a DPEase activity of about 40.0 U/mL. 
     These results prove the interest of using a fermentation medium comprising at least 60 g/L of sugar, notably glucose. 
     Example 5: Comparison of Several Mutated Nucleotide Sequences of 5′UTR 
     Mutations have been brought in the nucleotide sequences of the 5′ untranslated region upstream of the ATG codon of the DPEase gene. 
     Results of the DPEase activity, tested according to the Standard Of Procedure (SOP), is detailed in Table 7 below. 
     
       
         
           
               
             
               
                 TABLE 7 
               
             
            
               
                   
               
               
                 DPEase activity of several variants 
               
            
           
           
               
               
               
               
               
            
               
                   
                 nt upstream of 
                   
                   
                   
               
               
                 clone # 
                 start codon 
                 U/ml 
                 U/ml 
                 U/ml 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 original 
                 AGAGAGGAATGTACAC 
                 13.92 
                 13.92 
                 12.49 
               
               
                   
                 (SEQ ID NO: 41) 
                   
                   
                   
               
               
                   
               
               
                 I7 
                 GAAAGGAGGATTCGAA 
                 58.44 
                 58.44 
                 62.87 
               
               
                   
                 (SEQ ID NO: 42) 
                   
                   
                   
               
               
                   
               
               
                 I9 
                 GAAAGGAGGATTATGG 
                 77.4 
                 77.4 
                 81.51 
               
               
                   
                 (SEQ ID NO: 43) 
                   
                   
                   
               
               
                   
               
               
                 I11 
                 GAAAGGAGGATTGTCG 
                 21.81 
                 21.81 
                 22.29 
               
               
                   
                 (SEQ ID NO: 44) 
                   
                   
                   
               
               
                   
               
               
                 II2 
                 GAAAGGAGGATTTAGT 
                 55.72 
                 55.72 
                 57.39 
               
               
                   
                 (SEQ ID NO: 45) 
                   
                   
                   
               
               
                   
               
               
                 II3 
                 GAAAGGAGGATTGAGG 
                 55.91 
                 55.91 
                 55.67 
               
               
                   
                 (SEQ ID NO: 46) 
                   
                   
                   
               
               
                   
               
               
                 II6 
                 AGAAAGGAGGATTAAA 
                 73.25 
                 73.25 
                 75.43 
               
               
                   
                 (SEQ ID NO: 47) 
                   
                   
                   
               
               
                   
               
               
                 II7 
                 GAAAGGAGGATTTCGT 
                 75.45 
                 75.45 
                 80.24 
               
               
                   
                 (SEQ ID NO: 48) 
                   
                   
                   
               
               
                   
               
               
                 II8 
                 GAAAGGAGGATTTTTG 
                 49.79 
                 49.79 
                 51.95 
               
               
                   
                 (SEQ ID NO: 49) 
               
               
                   
               
            
           
         
       
     
     Clones 116 and 117 provides the best DPEase activity after analysis according to SOP. However, assays under optimal fermentation conditions (see example 4) showed that mutations of the 17 clone lead to the best DPEase activity. 
     Thus, mutations of the 17 clone are the mutations present in the plasmid pR3.