Patent Application: US-201515113978-A

Abstract:
pmxa synthetase involved in polymyxin e synthesis , comprising four adenylation sites , characterized in that the second adenylation site has at least 90 % identity with the peptide sequence seq id no 1 : vteaekadllgrfndtttefprgktliqlfeeqveripdaaaitlneqe ltyrelnervnrlartlrshgiskgrlvailaersiemvvgmlaahkag aayvpidpeypeerirfliedsggqvmltqsrlrerlagsdpvilldde sfyhedgtnlntgieatdlacviytsgttgkpkgnpvshrnivrvvqnt nyiditerdhvlqlssysf dg at f difgaltngarlvlvpyetlleigr ladliqrerisvm f ittaffnilvdvnvdclrdvrai l f g gervsvghv rkalahigpgrlnh v ygptest vy ttylpvdfvdelavtvpigrpisnt tvyivdsrnkllpigvagelcvggeglvrgynnrpeltaekfvdnpfvp germyrtgdlakwlpdgtieyvgrtddqvkirgfrielgeieaqlqkve girkttvfarenasgekqlcayyeadcelpaaelksvlskelpaymipa yliqlerlplttng k vdrrslpapeeslqpggg , the underlined amino acids dgfflgvvyk being conserved and forming a binding pocket specific for a leucine , isoleucine or valine residue .

Description:
the invention relates to the isolation and identification of new genes encoding polymyxin e synthetases , in particular of a gene encoding the “ pmxa ” synthetase having a particular adenylation site , forming a specific binding pocket allowing the integration into the polymyxin molecule of a leucine or isoleucine residue . all the technical terms used in the present application are well known to the person skilled in the art and are defined in the reference book by sambrook et al . “ molecular cloning : a laboratory manual ”. the term “ polynucleotide ” denotes a chain of covalently bonded nucleotides . for the purposes of the invention , this term denotes nucleic acids such as ribonucleic acid ( rna ) and deoxyribonucleic acid ( dna ). for the purposes of the present invention , the “ percentage identity ” between two nucleic acid sequences is determined by comparing the two optimally aligned sequences through a comparison window . the part of the nucleotide sequence in the comparison window may comprise additions or deletions ( for example gaps ) compared with the reference sequence ( which does not comprise these additions or these deletions ) so as to obtain optimal alignment between the two sequences . the percentage identity is calculated by determining the number of positions at which an identical nucleic base is observed for the two sequences compared , then by dividing the number of positions at which there is identity between the two nucleic bases by the total number of positions in the comparison window , then by multiplying the result by one hundred in order to obtain the percentage nucleotide identity of the two sequences with respect to one another . the optical alignment of the sequences for the comparison may be carried out by a computer using known algorithms . entirely preferably , the percentage sequence identity is determined using the clustal w software ( version 1 . 82 ), the parameters being fixed as follows : ( 1 ) cpu mode = clustalw mp ; ( 2 ) alignment =“ full ”; ( 3 ) output format =“ aln w / numbers ”; ( 4 ) output order =“ aligned ”; ( 5 ) color alignment =“ no ”; ( 6 ) ktup ( word size )=“ default ”; ( 7 ) window length =“ default ”; ( 8 ) score type =“ percent ”; ( 9 ) topdiag =“ default ”; ( 10 ) pairgap =“ default ”; ( 11 ) phylogenetic tree / tree type =“ none ”; ( 12 ) matrix =“ default ”; ( 13 ) gap open =“ default ”; ( 14 ) end gaps =“ default ”; ( 15 ) gap extension =“ default ”; ( 16 ) gap distances =“ default ”; ( 17 ) tree type =“ cladogram ” and ( 18 ) tree gap distances =“ hide ”. the term “ polypeptide ” denotes a chain of covalently bonded amino acids . the term “ polymyxin e synthetases ” denotes the enzymes capable of integrating a specific amino acid into the chain of amino acids forming polymyxin e , and where appropriate of converting the amino acid so as to form a cyclic structure . the term “ adenylation site ” or “ adenylation domain ” denotes , in the polymyxin synthetase molecule , the domain which plays a role in the choice and activation of the amino acid , while the “ condensation domain ” catalyzes the formation of a peptide bond and the “ thiolation domain ” is responsible for the transport of the compounds undergoing formation between the modules . the term “ epimerization site ” denotes , in a synthetase , a domain which makes it possible to convert a residue which has a levorotary chirality “ l ” into its enantiomer of dextrorotary chirality “ d ”, in particular an l - α , γ - diaminobutyric acid ( l - dab ) into a d - α , γ - diaminobutyric acid ( d - dab ). the term “ binding pocket ” denotes a zone of the protein which has a three - dimensional structure in the form of a “ pocket ”, in which high - specificity interactions with a substrate or , in the present case , an amino acid , will make it possible to select and activate this amino acid . the expression “ the underlined amino acids being conserved ” means that , even if slight sequence variations may be observed between two proteins having the same catalytic activity , the amino acids indicated are essential to the specificity and to the activity of this protein and may not therefore be modified , without risk of losing or reducing the specificity and / or the activity of this protein . the present invention relates to a pmxa synthetase involved in polymyxin e synthesis , comprising four adenylation sites , characterized in that the second adenylation site has at least 90 % identity with the following peptide sequence : the underlined amino acids dgfflgvvyk being conserved and forming a binding pocket specific for a leucine , isoleucine or valine residue . according to one particular aspect of the invention , the second adenylation site of pmxa has 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 % or 100 % identity with the peptide sequence seq id no 1 , the underlined amino acids dgfflgvvyk being conserved and forming a binding pocket specific for a leucine , isoleucine or valine residue . according to one particular aspect of the invention , the second adenylation site of pmxa has a peptide sequence comprising or consisting of the peptide sequence seq id no 27 , the underlined amino acids dgfflgvvyk being conserved and forming a binding pocket specific for a leucine , isoleucine or valine residue . according to one particular aspect of the invention , the pmxa synthetase comprises four specific binding pockets comprising the following amino acids : dawivgaivk ( seq id no 2 ), specific for a leucine residue ; dgfflgvvyk ( seq id no 3 ), specific for a leucine , isoleucine or valine residue ; dvgeisaidk ( seq id no 4 ), specific for a diaminobutyric acid residue ; dvgeisaidk ( seq id no 5 ), specific for a diaminobutyric acid residue . it is clearly understood that these sequences seq id no 2 , 3 , 4 and 5 group together the essential amino acids forming a functional binding pocket in a three - dimensional structure , but are not consecutive amino acids in a peptide sequence . according to one particular aspect of the invention , the pmxa synthetase comprises the polypeptide sequence presented in seq id no 6 , and shown in fig5 , the adenylation domains being indicated in bold , the amino acids forming the binding pocket being underlined . in particular , this sequence may comprise , in addition to the sequence presented in seq id no 6 , additional amino acids at the n - and c - terminal ends . according to one particular aspect , the polypeptide sequence of the pmxa synthetase consists of the polypeptide sequence presented in seq id no 6 . according to another aspect of the invention , the pmxa synthetase has the sequence which comprises , or consists of , the peptide sequence presented in seq id no 25 . the pmxa synthetase has four adenylation sites at the following positions : from residue 234l to residue 751y ; from residue 1741v to residue 2263g ; from residue 2815p to residue 3345t ; from residue 3900e to residue 4428g . it is understood that these positions are identified according to a particular peptide sequence , in this case according to seq id no 6 comprising 4967 amino acids , and may be redefined for sequences of larger size . in particular , when the pmxa synthetase has a sequence consisting of the sequence seq id no 25 , the four adenylation sites are in the following positions : from residue 265l to residue 782y ; from residue 1772v to residue 2294g ; from residue 2846p to residue 3376t ; from residue 3931e to residue 4459g . the present invention also relates to a pmxe synthetase involved in polymyxin e synthesis , comprising five adenylation sites and one epimerization site , characterized in that the epimerization site has at least 90 % identity with the peptide sequence as presented in seq id no 30 . according to one particular aspect of the invention , the peptide sequence of the pmxe synthetase comprises or consists of the sequence represented in sequence seq id no 14 . this synthetase is responsible for the integration of residues 1 to 5 ( see fig1 for the numbering ) into the peptide chain of the polymyxin e molecule . this synthetase has the functional characteristic of being able to convert the l - dab residue into its stereoisomer d - dab , and of incorporating said stereoisomer in position 3 in a polymyxin e peptide chain . such a variant polymyxin molecule might have improved antimicrobial properties , as has been proposed in the literature ( hong s y et al ., 1999 ; lee d l et al ., 2004 ). the present invention also relates to a nucleic acid comprising an open reading frame , encoding a synthetase involved in polymyxin e synthesis , having at least 90 % identity with the nucleotide sequence seq id no 7 , with the proviso that the nucleotides encoding the underlined amino acids dgfflgvvyk of the sequence seq id no 1 are conserved . according to one particular aspect of the invention , the nucleic acid encoding a synthetase involved in polymyxin e synthesis has 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 % or 100 % identity with the nucleotide sequence seq id no 7 , with the proviso that the nucleotides encoding the underlined amino acids dgfflgvvyk of the sequence seq id no 1 are conserved . the expression “ the nucleotides [ . . . ] are conserved ” indicate that these nucleotides encoding the amino acids dgfflgvvyk of the sequence seq id no 1 must be either identical to those observed in the same position in the sequence seq id no 7 , or different but with the proviso that the codons formed by these nucleotides encode the amino acids dgfflgvvyk underlined in the sequence seq id no 1 . this is because , and as indicated above , these amino acids are essential to the specificity and to the activity of this synthetase and may not therefore be modified , without the risk of losing or reducing the specificity of this binding pocket . according to another particular aspect of the invention , the nucleic acid encoding a pmxa synthetase involved in polymyxin e synthesis comprises a nucleotide sequence encoding the second adenylation site of this pmxa synthetase , consisting of the nucleotide sequence presented in sequence seq id no 28 . this sequence encodes the peptide sequence represented in seq id no 27 and also encodes the peptide sequence represented in seq id no 1 which has two amino acids fewer than seq id no 27 at the c - terminal end of the domain . the present invention also relates to a group of genes encoding enzymes involved in polymyxin e synthesis , comprising in particular : a gene encoding the pmxa synthetase as defined above , and genes encoding respectively the pmxb and pmxe synthetases . according to one preferred aspect of the invention , this group of genes comprises the pmxe gene encoding a pmxe synthetase comprising an epimerization domain , the peptide sequence of which comprises or consists of the sequence represented in seq id no 30 . according to one preferred aspect of the invention , this group of genes also comprises genes encoding respectively the pmxc and pmxd transport proteins . the sequences of the pmxc and pmxd proteins isolated from the paenibacillus alvei strain are shown in sequences seq id no 12 and no 13 , respectively . according to another particular aspect of the invention , this group of genes involved in polymyxin e synthesis comprises : a pmxa gene having at least 90 % identity with the sequence seq id no 7 , with the proviso that the nucleotides encoding the underlined amino acids dgfflgvvyk of the sequence seq id no 1 are conserved , a pmxb gene having at least 90 % identity with the sequence seq id no 8 , and a pmxe gene having at least 90 % identity with the sequence seq id no 9 . according to another aspect of the invention , this group of genes involved in polymyxin e synthesis comprises : a pmxa gene having at least 95 % identity with the sequence seq id no 7 , with the proviso that the portion of nucleic acid encoding the underlined amino acids dgfflgvvyk is conserved , a pmxb gene having at least 95 % identity with the sequence seq id no 8 , and a pmxe gene having at least 95 % identity with the sequence seq id no 9 . according to another aspect of the invention , this group of genes involved in polymyxin e synthesis comprises : a pmxa gene comprising a nucleotide sequence having 100 % identity with the sequence seq id no 7 , and in particular comprising a nucleotide sequence as presented in seq id no 26 , a pmxb gene comprising a nucleotide sequence having 100 % identity with the sequence seq id no 8 , and a pmxe gene comprising a nucleotide sequence having 100 % identity with the sequence seq id no 9 . the present invention also relates to a nucleic acid comprising or consisting of a sequence seq id no 10 , or a sequence seq id no 29 , representing the complete sequence of the cluster derived from the paenibacillus alvei strain , comprising the pmxa , pmxb , pmxc , pmxd and pmxe genes involved in polymyxin e synthesis . table 1 below presents the various nucleic acid and protein sequences referenced in the present application : the present invention also relates to an expression vector comprising a gene encoding a pmxa synthetase as defined above , or a nucleic acid encoding a pmxa synthetase as defined above , and / or a gene encoding a pmxe synthetase as defined above . in the context of the invention , the terms “ vector ”, “ expression vector ” and “ plasmid ” are equivalent and are used in accordance with the usual acceptance in the molecular biology , genetic engineering and microbiology field . very briefly , it is a non - viral dna molecule housed by a host cell , distinct from the natural chromosomal dna of said host cell and capable of autonomous replication . a “ vector ” is obtained by conventional molecular biology and genetic engineering techniques , and is a molecule into which one or more exogenous nucleotide sequences have been inserted ( or cloned ). the present invention relates to a vector for the expression , by a host cell , of exogenous nucleotide sequences , comprising : an origin of replication , elements allowing the expression of the genes introduced , such as an appropriate promoter , enhancers , etc ., at least one nucleotide sequence of which the expression is desired . the choice of the vector and , more particularly , of the origin of replication that it carries depends on the host cell that will house it . depending on the type of host cells , several copies of a vector and / or several different vectors may be introduced into the same host cell , simultaneously or sequentially . the choice of the vector will also depend on the size of the nucleic acid sequence to be expressed . in particular , for sequences greater than 20 kilobase pairs , fosmids , which are vectors capable of containing large nucleic acid sequences ( up to 40 kilobase pairs ), will be preferred . it is also possible to use several vectors , each comprising a portion of a set of genes , and to transfer several vectors into the same host cell , this making it possible to express an entire set of genes in the same strain . other vectors that may contain sequences of large sizes are cosmids and bacterial artificial chromosomes ( bacs ). according to one preferred aspect of the invention , the expression vector totally or partly comprises a group of genes as defined above . according to another preferred aspect of the invention , the expression vector totally or partly comprises a nucleic acid as defined above , in particular comprising the sequence presented in seq id no 10 or in seq id no 29 . preferably , the expression vector is a fosmid comprising all of the dna sequence encoding the complete cluster comprising the pmxa , pmxb , pmxc , pmxd and pmxe genes derived from paenibacillus alvei , having the sequence seq id no 10 . other enzymes are involved in polymyxin synthesis , in particular polymyxin e synthesis . these are enzymes which allow the biosynthesis of the dab residue , this residue being the residue predominantly integrated into the peptide chain of polymyxin e , and enzymes which allow the bonding of the fatty acid on the peptide part . an enzyme for biosynthesis of the α , γ - diaminobutyric acid ( dab ) residue has been identified and isolated from the bacterial strain of paenibacillus alvei described in the examples . in particular , the peptide sequence of this enzyme comprises or consists of the peptide sequence presented in sequence seq id no 31 . the nucleic acid encoding this enzyme comprises or consists of the nucleotide sequence presented in sequence seq id no 32 . preferably , this enzyme with transaminase activity may be expressed in a microorganism intended to produce polymyxin e , in combination with the pmxa , pmxb and pmxe synthetases , and preferably also with the pmxc and pmxd transport molecules . this transaminase comprising 431 residues has a sequence homology with enzymes exhibiting similar functions , derived from other microorganisms , as is shown in table 2 below : enzymes which allow the bonding of the fatty acid on the peptide chain of polymyxin e , said enzymes therefore having acyl transferase activity , have also been identified and isolated from the bacterial strain of paenibacillus alvei described above . preferably , at least one acyl transferase will be expressed in a microorganism intended for producing polymyxin e , in combination with the pmxa , pmxb and pmxe synthetases , and preferably also with the pmxc and pmxd transport molecules , and more preferably also with an enzyme for the biosynthesis of the α , γ - diaminobutyric acid ( dab ) residue . alternatively , the bonding of the fatty acid on the peptide chain of polymyxin e may also be carried out by chemical coupling , according to one of the techniques well known to those skilled in the art . the term “ host cell ” or “ host microorganism ” is used in the context of the present invention to denote a cell which has been transformed , i . e . into which the exogenous dna has been introduced , this exogenous dna being in particular in the form of an expression vector comprising a sequence of interest , which will be expressed by means of the cellular machinery of the host cell , capable of synthesizing , from the exogenous dna , the messenger rnas and the proteins corresponding to this dna . the present application relates in particular to a microorganism transformed by introducing a gene or group of genes as defined above , or one or more expression vectors as presented above . the invention relates in particular to a microorganism transferred by introducing : a gene encoding a pmxa synthetase as defined above , or a gene encoding a pmxe synthetase as defined above , or an expression vector comprising a dna sequence encoding a pmxa synthetase as defined above , in particular the sequence as presented in seq id no 7 or in seq id no 26 . the invention also relates to a microorganism transformed by introducing a group of genes as defined above , or a nucleic acid encoding all or part of the pmxa , pmxb and pmxe genes , as defined above , or at least one expression vector comprising a gene encoding all or part of the pmxa , pmxb and pmxe genes , as defined above . according to one particular aspect of the invention , the microorganism is in addition transformed so as to comprise at least one nucleic acid encoding an enzyme involved in the biosynthesis of the dab residue , and in particular a nucleic acid comprising a sequence comprising or consisting of the sequence seq id no 32 . according to another aspect of the invention , the microorganism is in addition transformed so as to comprise at least one nucleic acid encoding an enzyme with acyl transferase activity , catalyzing the bonding of a fatty acid on the peptide chain of polymyxin e . the transformation of microorganisms is a technique commonly used in molecular biology laboratories , which makes it possible to introduce exogenous dna into the microorganism . various techniques enable the transformation of a microorganism , and in particular of a competent bacterium , and are all well known to those skilled in the art . mention may in particular be made of electroporation , and the use of calcium chloride followed by a heat shock . in one preferred aspect of the invention , in the transformed microorganism , the gene or the group of genes introduced is overexpressed . the overexpression of a gene may be defined by an increased expression of this gene , i . e . a greater production of messenger rna and the protein encoded by this gene , in the cell . the expression of the protein will in particular be increased by 50 %, by 100 %, by 150 %, by 200 %, or even by 300 % compared with the level of endogenous expression observed in a bacterium expressing this gene naturally , before any transformation . the overexpression of a gene may be obtained in several ways , all well known to those skilled in the art . mention will in particular be made of : using strong promoters for controlling the level of gene expression and increasing the number of copies of the gene in the cell . preferably , the vector used will comprise a strong promoter under the control of which the gene will be inserted , and said vector will be present in a high number of copies , in particular 10 , 20 , 50 or 100 copies in the host cell . those skilled in the art will know how to choose the host microorganism most suitable for the expression of a vector comprising the gene ( s ) according to the invention . in particular , gram - positive bacteria will be preferred . a microorganism which is particularly suitable is a bacterium belonging to the bacillus or paenibacillus genus . the species b . subtilis and paenibacillus polymyxa are particularly suitable for the expression of polymyxin synthetases and the production of polymyxin e . the strains of the paenibacillus genus , and in particular of paenibacillus alvei , and more particularly the paenibacillus alvei strain isolated from the environment by the inventors , may also be used as host microorganism to be transformed with the vector or the nucleic acid as defined above . finally , synthetic bacteria may also be used in the context of the present invention , as host cells . the present application also relates to a method for producing polymyxin e , comprising : culturing a transformed microorganism as defined above , in an appropriate mineral medium , and purifying , from the culture medium , the polymyxin e produced . the term “ appropriate mineral medium ” denotes a culture medium which allows the growth of microorganisms , and which comprises in particular mineral salts and nutrients . a preferred medium has the following specific composition : 0 . 45 % ( w / v ) anhydrous kh 2 po 4 , 1 . 13 % ( w / v ) k 2 hpo 4 . 3h 2 o , 0 . 6 % ( w / v ) ( nh 4 ) 2 so 4 , 0 . 6 % ( w / v ) glucose , 0 . 001 % 0 ( w / v ) thiamine , 0 . 02 % ( w / v ) mgso 4 . 7h 2 o . this medium may also comprise , where appropriate , a precursor required for polymyxin synthesis , in particular diaminobutyric acid . according to one preferred aspect of the invention , the culturing is carried out at a temperature of 30 ° c ., and lasts at least 25 hours . preferably , the culturing takes place in 200 ml of mineral medium in 1 l flasks , with shaking , for 30 h at 30 ° c . the present invention also relates to a method for producing polymyxin e variants , comprising culturing a microorganism as defined above , in an appropriate mineral medium , and purifying , from the culture medium , the polymyxin e variant ( s ). the present invention also relates to polymyxin e variants obtained according to the production method as described above . the “ polymyxin e variants ” denote molecules derived from the structure of polymyxin e ( see fig1 ) and which may for example have one or more different amino acids , without however being classified as belonging to another type of polymyxin . in particular , the types of molecules detected in the culture medium for the paenibacillus alvei strain bl - r are polymyxin e variants which have the following structural difference : the residue in position 3 may be a d - dab residue in place of an l - dab residue observed in the conventional structure of polymyxin e . the variant forms may be natural or synthetic . the term “ natural variants ” is intended to mean variants synthesized by non - transformed microorganisms ; the term “ synthetic variants ” is intended to mean variant forms synthesized by transformed microorganisms , which have not been identified in the natural state . in particular , the polymyxin synthetases encoded by the pmxa , pmxb and pmxe genes may be modified so as to become specific for the binding of certain amino acids which are part of the composition of polymyxin e , in order to synthesize polymyxin e variants which have fewer cationic charges and which are therefore less toxic to the human organism . these polymyxin e variants have advantages and in particular a lower toxicity than polymyxin e . moreover , the clinical use of various polymyxin e variants makes it possible to reduce the appearance of antiobiotic resistance . it is probable that , for the variants comprising d - dab residues in place of l - dab residues , the antimicrobial activity of these molecules is increased compared with the antimicrobial properties observed with the conventional polymyxins . the present invention relates to these variants , and also to the use thereof in the treatment of bacterial infections with gram - negative bacteria . the examples below illustrate the invention claimed , but are not limiting . a microorganism ( b - lr ) producing colistin ( polymyxins e1 and e2 ), belonging to the paenibacillus alvei species , was isolated from the environment and cultured . a genomic dna library was constructed in escherichia coli using fosmid vectors ( 900 clones ). the search for the genes encoding the enzymes which synthesize colistin was carried out by degenerate pcrs . the primers used were defined with the aim of amplifying the domains specializing in the integration of diaminobutyric acid ( dab ), which is the amino acid most representing the structure of colistin ( 6 amino acids / 12 ). three clones of interest were selected from the sequence ( roche gs flx ). the sequences obtained could be assembled over 50 kb . the open reading frames were sought . five of them called a , b , c , d and e describe a cluster of approximately 41 kb with a ( 14 . 9 kb ), b ( 3 . 3 kb ), c ( 1 . 8 kb ), d ( 1 . 7 kb ) and e ( 18 . 9 kb ), which is represented in fig2 . the a , b and e genes were identified , by sequence homology , as encoding synthetases . an in silico study made it possible to predict the involvement of these synthetases in the assembly of colistin ( http :// nrps . informatik . uni - tuebingen . de / and http :// nrps . igs . umaryland . edu / nrps /). the c and d genes could , for their part , be involved in the export and resistance of the producer microorganism with respect to colistin . these oligonucleotides are represented in seq id nos 15 to 24 . comparison of the adenylation domains of various paenibacillus strains producing molecules belonging to the polymyxin family the enzymes involved in polymyxin production belong to the non - ribosome synthetase ( nrps ) family . these synthetases are capable of producing a particular peptide without relying on the translation of an mrna template . the specificity of the peptide chain produced depends on a precise sequence of amino acids of the synthetase constituting an adenylation domain . four adenylation domains were identified in silico for the synthetase a of b - lr , one for b and five for e . each adenylation domain contains a signature of ten amino acids which confers it on the specificity of integrating a precise amino acid into the non - ribosomal peptide during elongation . these signatures were identified in silico and compared to those described in the literature ( table 4 ) using the program available at the following web address : http :// nrps . informatik . uni - tuebingen . de /. the e681 and atcc21830 strains are described in patent u . s . pat . no . 8 , 329 , 430 . the pkb1 strain is described in the article by shaheen et al ., 2011 . the m - 1 strain is described in the article by niu et al ., 2013 . the synthetases of b - lr differ at the level of the second adenylation domain of the pmxa synthetase and of the third domain of the pmxe synthetase . the comparisons with the protein sequences derived from the bl - r strain with the protein sequences already known are presented in table 5 below : a sequence of 1650 bp beginning at the end of pmxd and finishing at the start of pmxe was selected and amplified . it has the particularity of possessing a unique sacii enzyme restriction site . the amplified sequence was cloned into the pgem ® 7z plasmid . the gene encoding apramycin resistance was integrated at the sacii restriction site . this construct was introduced into b - lr by electroporation . the selection of the mutants having undergone a double recombination event was carried out by culturing on agar medium supplemented with apramycin . the antiomicrobial activity of the supernatant of the mutants was compared with that of b - lr . the culture supernatant of mutant 5 is less active than that of the wild - type b - lr bacterium on p . aeruginosa ( fig4 ). the peptide sequence of the pmxe synthetase is presented in sequence seq id no 14 . the sequence of the epimerization domain is presented in seq id no 30 ; it comprises at least 461 residues comprised between and including the residues val 3205 to leu 3665 of the sequence seq id no 14 . one of the characteristics of non - ribosomal peptides is the presence of amino acids with ( l ) stereoisomerism . usually , an ( l ) amino acid is activated by the a domain , which then incorporates it directly . however , sometimes , this amino acid of the ( l ) series may be converted into its ( d ) form by any epimerization domain , before the formation of the peptide bond . according to the in silico predictions , two epimerization domains were identified in modules 3 and 6 of the pmx cluster . this suggests that a stereochemical change in the amino acids present at these sites is possible . thus , the amino acids present in position 3 ( dab - 3 ) and in position 6 ( leu - 6 ) would be incorporated into the peptide chain in their ( d ) form . this was known for leu - 6 , incorporated in its ( d ) form into all polymyxins e , but is unusual for the dab - 3 residue . the pmxe synthetase according to the invention therefore has the property of converting the l - dab residue into its stereoisomer ( d ), and of integrating it in position 3 ( see fig1 ) in the polymyxin e molecule synthesized . said polymyxin e molecules comprising a d - dab residue in position 3 are polymyxin e variants . production of polymyxin e by a microorganism expressing the combination of a pmxa , a pmxb and a pmxe characterized in the preceding examples the microorganism ( b - lr ) produces several types of polymyxin e molecules comprising a leucine , isoleucine or valine residue in position 7 . these various types of molecules could be detected in the culture medium by mass spectrometry , according to the protocol detailed below : a control solution of colistin sulfate at 0 . 0002 % was prepared in the microorganism culture medium ( m63t medium ). four fractions were recovered and analyzed by uplc - ms ( ultra performance liquid chromatography — tandem mass spectrometry ). the two peaks corresponding to the polymyxins e1 and e2 present in the positive control were found in the fourth extraction fraction . the b - lr strain exhibits a maximum level of antimicrobial activity at 30 hours of culture . at this time , the culture supernatant was recovered and filtered ( 0 . 22 μm ) before being subjected to a solid - phase extraction step . the peptides of the fourth fraction were analyzed by uplc - ms . seventeen elution peaks were observed in this fourth fraction . among them , the main peaks exhibiting retention times of 1 . 61 and 1 . 52 minutes have [ m + h ] + molecular masses respectively of 1169 . 7727 and 1155 . 7555 . they correspond respectively to the known polymyxins e1 and e2 , which have the same peptide structure and differ by their fatty acids , respectively of formulae c 9 h 17 o and c 8 h 15 o . ms / ms analyses were carried out in order to confirm these structures , as have previously been presented in the articles by govaerts et al ., 2002 ; and by decrescenzo et al ., 2007 . tandem mass spectrometry ( ms / ms ) is used to determine the series of amino acids through fragmentation of the peptide bond . this technique consists in combining two analyzers separated by a collision cell . a first step consists in selecting an ion derived from the source of ionization in the quadrupole : this is the parent or precursor ion . this ion undergoes fragmentation in the collision cell under the effect of a bombardment by argon atoms , and gives “ daughter ” or “ product ” ions that will be analyzed and protected by the time - of - flight ( tof ) analyzer . the tof analyzer is based on the measurement of the time taken by the ions to cover a given distance , thereby making it possible to detect the m / z ratios and thus to determine the mass of the corresponding ions . during these experiments , the leucine and isoleucine residues cannot be differentiated , because of their identical molecular masses . three decapeptides with a ring containing seven amino acids were purified from the b - lr supernatant . their molecular weights range from 1140 . 74 da to 1168 . 77 da . the comparison of these molecules shows homologies at the level of residues 1 to 6 and 8 to 10 and differences at the level of residue 7 and also at the level of the fatty acid . these three molecules correspond to polymyxins ( colistins ) e 1 , e 2 and to val - colistin e 2 . the deduced chemical formulae thereof are presented in table 6 below . the mass spectra obtained for colistin e 1 , colistin e 2 and val - colistin e 2 have respective [ m + 2h ] 2 + values of 585 . 39 , 578 . 38 and 571 . 38 . the fragmentation of the parent ions of colistins e 1 and e 2 give daughter ions of m / z 829 , 728 , 628 , 427 , 327 and 227 ( fig6 and 7 ) which are formed by loss of amino acid fragments . the fragmentation of the parent ion of val - colistin e 2 gives daughter ions of m / z 815 , 714 , 614 , 514 , 413 , 313 and 213 ( fig8 ) also formed by loss of amino acid fragments . these daughter ions obtained are identical to those described in the literature during the fragmentation of colistins e 1 , e 2 and val - e 2 ( govaerts et al ., 2002 ; decrescenzo et al ., 2007 ). colistins e 1 and e 2 have the same amino acid sequences with a molecular mass difference of 14 da which corresponds to the loss of a ch 2 at the level of the fatty acid . in conclusion , these results show that the b - lr strain produces colistin e 1 , colistin e 2 and valine - colistin e 2 ( val - e 2 ). heterologous expression of the pmxa , b and e genes derived from paenibacillus in bacillus subtilis and detection of colistin bacillus is a heterologous expression host that is phylogenetically close to b - lr . the fosmid manipulated to house all of the colistin production cluster is transferred into the recipient strain by electroporation . the transformed strains are selected on agar medium supplemented with an antibiotic . the colistin production is carried out in liquid medium supplemented with diaminobutyric acid ( synthesis precursor ). the colistin is detected after separation of the biomass from the culture supernatant . extractions make it possible to isolate the colistin before characterization thereof by mass spectrometry . sambrook et al . ‘ molecular cloning : a laboratory manual ’. second edition , 1989 , cold spring harbor lab ., cold spring harbor , n . y . niu b , vater j , rueckert c , blom j , lehmann m , ru j j , chen x h , wang q , borriss r . “ polymyxin p is the active principle in suppressing phytopathogenic erwinia spp . by the biocontrol rhizobacterium paenibacillus polymyxa m - 1 ”. bmc microbiol . 2013 jun . 18 ; 13 ( 1 ): 137 . choi s k , park s y , kim r , kim s b , lee c h , kim j f , park s h —“ identification of a polymyxin synthetase gene cluster of paenibacillus polymyxa and heterologous expression of the gene in bacillus subtilis ” j . bacteriol . may 2009 vol . 191 no . 10 - 3350 - 3358 m shaheen , j li , a c ross , j c vederas , s e jensen —“ paenibacillus polymyxa pkb 1 produces variants of polymyxin b - type antibiotics ”— chemistry & amp ; biology , volume 18 , issue 12 , 23 dec . 2011 , pages 1640 - 1648 martti vaara , timo vaara —“ structure - activity studies on novel polymyxin derivatives that carry only three positive charges ”— peptides , volume 31 , issue 12 , december 2010 , pages 2318 - 2321 govaerts , c ., orwa , j ., van schepdael , a ., roets , e . & amp ; hoogmartens , j . characterization of polypeptide antibiotics of the polymyxin series by liquid chromatography electrospray ionization ion trap tandem mass spectrometry .— j pept sci 8 , 45 - 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( 2002a ). govaerts , c ., orwa , j ., van schepdael , a ., roets , e . & amp ; hoogmartens , j . liquid chromatography - ion trap tandem mass spectrometry for the characterization of polypeptide antibiotics of the colistin series in commercial samples . j chromatogr a 976 , 65 - 78 . ( 2002b ). hong s y , oh j e , lee k h . effect of d - amino acid substitution on the stability , the secondary structure , and the activity of membrane - active peptide . biochem pharmacol . 1999 dec . 1 ; 58 ( 11 ): 1775 - 80 . lee d l , powers j p , pflegerl k , vasil m l , hancock r e , hodges r s . effects of single d - amino acid substitutions on disruption of beta - sheet structure and hydrophobicity in cyclic 14 - residue antimicrobial peptide analogs related to gramicidin s . j pept res . 2004 february ; 63 ( 2 ): 69 - 84 . decrescenzo henriksen , d . r . phillips and j . b . doran peterson . polymyxin e production by p . amylolyticus e . letters in applied microbiology , volume 45 , issue 5 , pages 491 - 496 , november 2007 .