WO2007083112A2 - Lantibiotic biosynthetic gene clusters from a. garbadinensis and a. liguriae - Google Patents

Lantibiotic biosynthetic gene clusters from a. garbadinensis and a. liguriae Download PDF

Info

Publication number
WO2007083112A2
WO2007083112A2 PCT/GB2007/000138 GB2007000138W WO2007083112A2 WO 2007083112 A2 WO2007083112 A2 WO 2007083112A2 GB 2007000138 W GB2007000138 W GB 2007000138W WO 2007083112 A2 WO2007083112 A2 WO 2007083112A2
Authority
WO
WIPO (PCT)
Prior art keywords
seq
variant
nucleic acid
encoding
sequence
Prior art date
Application number
PCT/GB2007/000138
Other languages
English (en)
French (fr)
Other versions
WO2007083112A3 (en
Inventor
Steven Boakes
Jesus Cortes Bargallo
Michael John Dawson
Original Assignee
Novacta Biosystems Limited
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority to US12/161,221 priority Critical patent/US7989416B2/en
Priority to AU2007206769A priority patent/AU2007206769B2/en
Application filed by Novacta Biosystems Limited filed Critical Novacta Biosystems Limited
Priority to JP2008550839A priority patent/JP5219837B2/ja
Priority to US13/705,010 priority patent/USRE45003E1/en
Priority to NZ569486A priority patent/NZ569486A/en
Priority to BRPI0706525-6A priority patent/BRPI0706525A2/pt
Priority to DK07704921.1T priority patent/DK1979375T3/da
Priority to ES07704921T priority patent/ES2378090T3/es
Priority to MX2008009047A priority patent/MX2008009047A/es
Priority to EP07704921A priority patent/EP1979375B9/en
Priority to CN2007800067480A priority patent/CN101389641B/zh
Priority to CA002637315A priority patent/CA2637315A1/en
Priority to PL07704921T priority patent/PL1979375T3/pl
Priority to AT07704921T priority patent/ATE534659T1/de
Publication of WO2007083112A2 publication Critical patent/WO2007083112A2/en
Publication of WO2007083112A3 publication Critical patent/WO2007083112A3/en
Priority to IL192446A priority patent/IL192446A0/en
Priority to US13/157,454 priority patent/US8465947B2/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/365—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Actinoplanes (G)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/045—Actinoplanes

Definitions

  • This invention relates to characterisation of the biosynthetic gene cluster for the lantibiotic actagardine, identification of a novel variant of actagardine and its biosynthetic cluster, and methods of production and use of actagardine, a novel actagardine variant produced in a strain of A. liguriae, and variants of both of these produced according to this invention, utilizing genes from the characterised biosynthetic gene clusters.
  • Lantibiotics are peptides having antibiotic and other activities, produced by Gram-positive bacteria. They contain, among other modified residues, the thioether amino acids lanthionine and methyllanthionine, which cross- link the peptide chain into a polycyclic structure. They have been classified into two classes, type-A and type-B, though such classification is not unproblematic.
  • Type-A lantibiotics are generally elongate amphiphiles that are capable of forming pores in bacterial and other plasma membranes. Examples are nisin and subtilin.
  • Type-B lantibiotics by contrast, are globular, conformationally defined peptides that inhibit enzyme functions. Examples are cinnamycin and duramycin.
  • Activities ascribed to type-B lantibiotics such as cinnamycin include antimicrobial activity (providing potential application as antibiotics), inhibition of angiotensin-converting enzyme (providing a potential application in blood pressure regulation), immunomodulation via inhibition of phospholipase A2 (providing a potential application as anti-inflammatories), and interference with prostaglandin and leucotriene biosynthesis.
  • Type-B lantibiotics appear to exert their activity by interfering with enzyme activities by blocking the respective substrates.
  • type B lantibiotics such as mersacidin and actagardine have been found to inhibit biosynthesis of peptidoglycan; transglycosylation was identified as the target reaction.
  • the substrate for this reaction is the lipid-bound cell wall precursor lipid II. While this is a target for the lantibiotic vancomycin, the site of action is different and is a new target binding site not used by any current antibacterial drug.
  • Lantibiotics have been shown to have efficacy and utility as food additives and antibacterial agents against Propionibacte ⁇ um acnes and problematic pathogens, e.g. methicillin-resistant Staphylococcus aureus (MRSA), which has or is developing resistance to many commonly used antibiotics, and Streptococcus pneumoniae.
  • MRSA methicillin-resistant Staphylococcus aureus
  • Actagardine is a known type B tetracyclic lantibiotic, 19 amino acids in length (1890 Da). It has potent activity against important Gram positive pathogens such as Staphylococcus aureus and Streptococcus pyogenes both in vitro and in in vivo animal models.
  • the structure of actagardine is shown in Figure 4. The compound is produced from a pre-pro-peptide, the C- terminal portion of which has the polypeptide sequence of SSGWVCTLTI ECGTVICAC (SEQ ID NO:4).
  • the polypeptide of SEQ ID NO:4 is modified by the following crosslinks, creating secondary and tertiary structure: CROSSLINK 1-6, Lanthionine (Ser-Cys); CROSSLINK 7-12, Beta-methyllanthionine (Thr-Cys); CROSSLINK 9-17, Beta-methyllanthionine (Thr-Cys); CROSSLINK 14-19, Beta-methyllanthionine sulfoxide (Thr-Cys).
  • Actagardine has been reported to be produced by two species of Actinoplanes; A. garbadinensis and A. liguriae. Also co-produced is an analogue in which the CROSSLINK 14- 19 is not oxidized i.e. it is a beta-methyllanthionine not betamethyllanthionine sulfoxide which is named herein deoxy-actagardine.
  • the present invention relates to the cloned, sequenced and elucidated structural and regulatory information relevant to the biosynthetic gene cluster for the type-B lantibiotic, actagardine, from Actinoplanes garbadinensis and A. liguriae.
  • actagardine B a novel form of actagardine is produced which we have termed actagardine B or, in the non-oxidised form, deoxy-actagardine B.
  • actagardine B a novel form of actagardine is produced which we have termed actagardine B or, in the non-oxidised form, deoxy-actagardine B.
  • actagardine B a novel form of actagardine is produced which we have termed actagardine B or, in the non-oxidised form, deoxy-actagardine B.
  • the present invention provides the novel actagardine B and variants thereof, including variants based on the primary polypeptide sequences of SEQ ID NO:2 and SEQ ID NO:3, as well as variants thereof.
  • the invention provides nucleic acids encoding actagardine B and its variants, sets of nucleic acids and variants thereof derived from the above-mentioned gene clusters, methods of making actagardine B and its variants, and methods of generating novel variants of actagardine B.
  • Figure 1 provides a map of the actagardine encoding and regulatory gene cluster isolated from A. garbadinensis.
  • Figure 2 provides a map of the encoding and regulatory gene cluster isolated from A. liguriae which encodes a novel variant of actagardine, herein referred to as actagardine B.
  • Figure 3 provides a schematic showing a method disclosed herein for generation of actagardine variants utilizing nucleic acid sequences isolated from A. garbadinensis or from A. liguriae.
  • Figure 4 is a representation of the primary structure of mature actagardine where X1-X2 represent VaI-IIe, Y is -S(O)- and Z is NH 2 .
  • "Deoxy-actagardine B” is the Val15Leu lle16Val variant with a non-oxidised methyllanthionine bridge between AbuS14 and AlaS19.
  • SEQ ID NO:1 is the primary polypeptide sequence of Actagardine B:
  • SEQ ID NO:2 is the primary polypeptide sequence of Actagardine B variant VV: SSGWVCTLTIECGTVVCAC.
  • SEQ ID NO:3 is the primary polypeptide sequence of Actagardine B variant LI
  • SEQ ID NO:4 is the primary polypeptide sequence of Actagardine:
  • SEQ ID NO:11 is the primary polypeptide sequence of Ala-Actagardine B:
  • SEQ ID NO:12 is the primary polypeptide sequence of Ala-Actagardine B variant VV:
  • SEQ ID NO:13 is the primary polypeptide sequence of Ala-Actagardine B variant LI ASSGWVCTLTIECGTLICAC.
  • SEQ ID NO:14 is the primary polypeptide sequence of Ala-Actagardine:
  • SEQ ID NO:212 is the primary polypeptide sequence of pre-pro-Actagardine B:
  • SEQ ID NO:22 is the primary polypeptide sequence of pre-pro-Actagardine B variant W:
  • SEQ ID NO:23 is the primary polypeptide sequence of pre-pro-Actagardine B variant LI MSALAIEKSWKDVDLRDGATSHPAGLGFGELTFEDLREDRTIYAASSGWVCTLTIECG
  • SEQ ID NO:119 is the primary polypeptide sequence of pre-pro-Actagardine:
  • SEQ ID NO:100 is the non-vector, A. garbadinensis-de ⁇ ved, nucleotide sequence of the cosmid CosAG14.
  • SEQ ID NOs:101 - 132 are the polypeptide sequences of the open reading frames orf1 - orf32 of SEQ ID NO:100 respectively.
  • SEQ ID NO:200 is the non-vector, A. liguriae-de ⁇ ved, nucleotide sequence of the cosmid CosAL02.
  • SEQ ID NOs:201 - 231 are the polypeptide sequences of the open reading frames orf1 - orf31 of SEQ ID NO:200 respectively.
  • SEQ ID NOs:300-312 are primer sequences described herein below.
  • the present invention relates to the gene clusters of SEQ ID NO:100 and SEQ ID NO:200 and the polypeptides encoded by these clusters and variants thereof.
  • the polypeptide of SEQ ID NO:119 is pre-pro-actagardine and the polypeptide of SEQ ID NO:212 is pre-pro-actagardine B.
  • the remaining polypeptides and their variants are referred to herein generically as "cluster polypeptides”.
  • Cluster polypeptides derived from SEQ ID NO:100 are referred to as "1xx polypeptides” and those derived from SEQ ID NO:200 are referred to as "2xx polypeptides”.
  • Polypeptides which are 100% identical in both sequence and length to a cluster polypeptide are referred to as "wild-type" polypeptides.
  • a cluster polypeptide derived from SEQ ID NO:100 or SEQ ID NO:200 may be wild type or variant.
  • a polypeptide may be in substantially isolated form. Isolated polypeptides of the invention will be those as defined above in isolated form, free or substantially free of material with which it is naturally associated such as other polypeptides with which it is found in the cell. For example, the polypeptides may of course be formulated with diluents or adjuvants and still for practical purposes be isolated.
  • a polypeptide of the invention may also be in a substantially purified form, in which case it will generally comprise the polypeptide in a preparation in which more than 90%, e.g. 95%, 98% or 99% of the polypeptide in the preparation is a polypeptide of the invention.
  • Lantibiotic Polypeptide and LantibioticA gene refers generically to a type B lantibiotic polypeptide or the gene encoding such a peptide.
  • reference to these includes reference to cinnamycin, mersacidin, actagardine and actagardine B and the genes encoding these products.
  • Reference to a lantibiotic producing host cell refers to any host cell which in its native form produces a LanA polypeptide, as further defined herein below.
  • a LanA polypeptide is a polypeptide with anti-microbial activity. Anti-microbial activity may be examined by determining the MIC value against a reference organism, e.g. Micrococcus luteus. A LanA polypeptide is considered to exhibit anti-microbial activity if it has a MIC value of less than or equal to 16-fold higher than that of actagardine against the same strain of the reference microorganism.
  • the A. garbadinensis LanA gene is referred to as actA and the A. liguriae LanA gene is referred to as LigA.
  • LanM polypeptide is a polypeptide derived from a Lantibiotic gene cluster which is a modification factor required for the conversion of a precursor polypeptide to a lantibiotic compound.
  • LanM polypeptides include those of SEQ ID NO:120 (ActM) or a variant thereof, SEQ ID NO:213 (LigM) or a variant thereof, a cinM polypeptide as defined in WO02/088367, a mrsM polypeptide as disclosed in Altena et al, 2000, or a homologous polypeptide from another gene cluster of a bacteria which produces a type B lantibiotic.
  • LanR polypeptide is a polypeptide derived from a Lantibiotic gene cluster which is a regulatory factor required for the regulation of production of a precursor polypeptide.
  • LanR polypeptides include those of SEQ ID NO:122 (ActR) or a variant thereof, SEQ ID NO:216 (LigR) or a variant thereof, a cinR1 polypeptide as defined in WO02/088367, a mrsR1 polypeptide as disclosed in Altena et al, 2000, or a homologous polypeptide from another gene cluster of a bacteria which produces a type B lantibiotic.
  • LanT polypeptide is a polypeptide derived from a Lantibiotic gene cluster which is a transporter factor required for the production of a precursor polypeptide to a lantibiotic compound.
  • LanT polypeptides include those of SEQ ID NO:123 (ActT) or a variant thereof, SEQ ID NO:214 (LigT) or a variant thereof, a cinT polypeptide as defined in WO02/088367, a mrsT polypeptide as disclosed in Altena et al, 2000, or a homologous polypeptide from another gene cluster of a bacteria which produces a type B lantibiotic.
  • LanO polypeptide is a polypeptide derived from a Lantibiotic gene cluster which is a factor believed to be involved in the oxidation of the deoxy-form of actagardine and compounds of the invention to actagardine or to compounds of the invention in which Y is - S(O)-.
  • LanO polypeptides include those of SEQ ID NO:122 (ActO) or a variant thereof, SEQ ID NO:215 (LigO) or a variant thereof, or a homologous polypeptide from another gene cluster of a bacteria which produces a type B lantibiotic.
  • the invention provides an isolated cluster polypeptide selected from any one of SEQ ID NOs: 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111 , 112, 113, 114, 115, 116, 117, 118, 120, 121 , 122, 123, 124, 125, 126, 127, 128, 129, 130, 131 , 132, 201 , 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230 and 231.
  • the invention provides a cluster polypeptide which is a variant of any of the above-mentioned sequences.
  • Cluster polypeptides of particular interest include 1xx and 2xx polypeptides which are LanM, LanR, LanT or LanO polypeptides.
  • a “variant”, in relation to a cluster polypeptide, denotes: any polypeptide having an amino acid sequence which is different from, but which shows significant amino acid sequence identity with, the amino acid sequence of a reference polypeptide (in this case any wild type cluster polypeptide), or a fragment of that polypeptide.
  • significant amino acid sequence identity is preferably at least 80%, more preferably 85%, 90% or 95%, still more preferably 98% or 99%.
  • a variant is preferably of a length which is the same as, or at least 70%, preferably at least 80%, more preferably at least 90% and most preferably at least 95% of the length of the wild type cluster polypeptide.
  • Percent (%) amino acid sequence identity is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the sequence with which it is being compared, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity.
  • the % identity values used herein are generated by BLAST- 2 which was obtained from Altschul et al. (1996); http://blast. wustl/edu/blast/README. html.
  • the HSPS and HSPS2 parameters are dynamic values and are established by the program itself depending upon the composition of the particular sequence and composition of the particular database against which the sequence of interest is being searched; however, the values may be adjusted to increase sensitivity.
  • a % amino acid sequence identity value is determined by the number of matching identical residues divided by the total number of residues of the "longer" sequence in the aligned region, multiplied by 100.
  • the "longer" sequence is the one having the most actual residues in the aligned region (gaps introduced by WU BLAST-2 to maximize the alignment score are ignored).
  • a variant will retain a biological function of the reference polypeptide.
  • biological function is retained wherein the variant, when present in a host cell with the other members of its cluster, is capable of producing a lantibiotic.
  • This may be determined, for example, by providing a host cell containing SEQ ID NO:100 in the case of a 1xx cluster polypeptide variant, or SEQ ID NO:200 in the case of a 2xx polypeptide variant, wherein the host cells produce actagardine or actagardine B respectively, modifying the sequence to encode the variant, and determining whether a lantibiotic polypeptide is still produced.
  • the invention provides polypeptides, preferably in isolated form, which are precursors of the compounds of the present invention or of actagardine.
  • the precursor polypeptides include the polypeptides of any one of SEQ ID NOs:1-4, SEQ ID NOs:11-14, SEQ ID NOs:212, 22, 23 and 119, as well as variants or derivatives thereof which can be converted to a lantibiotic polypeptide.
  • a variant of a precursor polypeptide of any one of SEQ ID NOs: 1-4 is a polypeptide in which one or more, for example from 1 to 5, such as 1 , 2, 3 or 4 amino acids are substituted by another amino acid.
  • the amino acid is at a position selected from positions 2, 3, 4, 5, 8, 10, 11 , 13 or 18 of any one of SEQ ID NOs:1-4.
  • a variant of a precursor polypeptide of any one of SEQ ID NOs:11-14 is a polypeptide in which one or more, for example from 1 to 5, such as 1, 2, 3 or 4 amino acids are substituted by another amino acid.
  • the amino acid is at a position selected from positions 3, 4, 5, 6, 9, 11 , 12, 14 or 19 of any one of SEQ ID NOs:11-14.
  • a variant of a precursor polypeptide of any one of SEQ ID NOs:212, 22, 23 and 119 is a polypeptide in which one or more, for example from 1 to 5, such as 1 , 2, 3 or 4 amino acids of the C-terminal region (residues 46-64) corresponding to SEQ ID NOs:1-4 respectively are substituted by another amino acid.
  • the amino acid is at a position selected from positions corresponding to positions 2, 3, 4, 5, 8, 10, 11 , 13 or 18 of any one of SEQ ID NOs:1-4.
  • Such variants may further include changes to the N-terminal region which retain at least 70%, for example at least 80%, preferably at least 90%, for example at least 95% of the N-terminal regions (residues 1-45).
  • a variant of the N-terminal region of SEQ ID NO:212 or SEQ ID NO:119 may comprise one or more substitutions (e.g. from 1 to 12, such as from 1 to 5, e.g. 1 , 2 or 3 substitutions at positions 4, 5, 6, 8, 9, 12, 13, 17, 18, 19, 21 and 32 which our data shows are varied between SEQ ID NO:212 and 119.
  • substitutions e.g. from 1 to 12, such as from 1 to 5, e.g. 1 , 2 or 3 substitutions at positions 4, 5, 6, 8, 9, 12, 13, 17, 18, 19, 21 and 32 which our data shows are varied between SEQ ID NO:212 and 119.
  • Substitutions may be of one amino acid by another naturally occurring amino acid and may be conservative or non-conservative substitutions.
  • Conservative substitutions include those set out in the following table, where amino acids on the same block in the second column and preferably in the same line in the third column may be substituted for each other:
  • substitutions may be of an amino acid which differs from the amino acid residue located in the corresponding location of SEQ ID NO:119, or vice versa. In either case, the substitution may be to introduce the SEQ ID NO:119 amino acid into SEQ ID NO:212, or vice versa (e.g. lie at position 4 of SEQ ID NO:212 may be substituted by Leu, and so on).
  • a precursor polypeptide may be obtained by expression of a nucleic acid encoding the polypeptide in a cell which is a non-producer of a lantibiotic.
  • the present invention provides a compound of the formula (I): wherein:
  • -X1-X2- represent -Leu-Val-; -VaI-VaI- or -Leu-lle-;
  • Y is -S- or -S(O)-; and Z is either H 2 N- or AIa-, or a pharmaceutically acceptable salt thereof.
  • the invention provides variants and biologically active derivatives of these compounds.
  • a variant of a compound of formula (I) is a compound which one of more, for example from 1 to 5, such as 1 , 2, 3 or 4 amino acids are substituted by another amino acid.
  • the amino acid is at a position selected from positions 2, 3, 4, 5, 8, 10, 11 , 13 or 18 of the compound of formula (I).
  • a variant may also comprise a substitution at position 15 or 16, provided that when both positions 15 and 16 are substituted and none of the other positions are changes, 15 and 16 are not VaI and lie respectively.
  • variants of compounds of the invention include those in which Ala- is replaced by another amino acid (particularly a naturally occurring amino acid encoded by the genetic code or its D- isoform), more particularly an amino acid selected from the group lie-, Lys-, Phe-, VaI-, GIu-, Asp-, His-, Leu, Arg-, Ser- and Trp-.
  • the amino acid may be selected from the group lie-, Lys-, Phe-, VaI-, GIu-, Asp-, His-, Leu-, Arg- and Ser-.
  • Derivatives of compounds of the invention are those in which one or more amino acid side chain of the compound of the invention has been modified, for example by esterification, amidation or oxidation.
  • Derivatives of compounds of the invention may be monoamide derivatives at one of the carboxy functions of actagardine, particularly at the C-terminal. More particularly, a derivative may be a compound in which the C-terminal of the compound of the invention is of the formula -COR, in which R represents the group -NR 1 R 2 , wherein R 1 and R 2 independently represent: (i) hydrogen; (ii) a group of formula -(CH 2 ) n -NR 3 R 4 , in which n represents an integer from 2 to 8 and
  • R 3 and R 4 independently represent hydrogen or (C 1 -C 4 ) alkyl or R 3 and R 4 taken together represent a group -(CHz) 3 -, -(CHz) 4 -, (CHa) 2 -O-(CHz) 2 -, -(CH 2 ) 2 -S-(CH 2 ) 2 - or -(CHz) 6 -; or R 1 and R 2 taken together with the adjacent nitrogen atom represent a piperazine moiety which may be substituted in position 4 with a substituent selected from: (a) (C 1 -C 4 )alkyl;
  • R represents the group -NR 1 R 2 , wherein R 1 and R 2 independently represent hydrogen, a group of formula -(CH 2 ) n - NR 3 R 4 , in which n represents an integer from 2 to 8 and R 3 and R 4 independently represent hydrogen or (Ci -C 4 ) alkyl or R 3 and R 4 taken together represent a group -(CH 2 ) 3 -, -(CH 2 ) 4 -, (CH 2 ) 2 -O-(CH 2 ) 2 -, -(CH 2 ) 2 -S- (CH 2 ) 2 - or -(CH 2 ) ⁇ -, or R 1 and R 2 taken together with the adjacent nitrogen atom represent a piperazine moiety which may be substituted in position 4 with a substituent selected from (C 1 - C 4 )alkyl, (C 5 -C 7 )-cycloalkyl, pyridyl, benzyl,
  • a derivative may include a compound in which the carboxy function of a side chain of an internal residue, e.g. that of the residue GIu11 , is modified from -COOH to a group -COOR 5 in which R 5 represents hydrogen, (C 1 -C 4 )alkyl or (C1 -C 4 )alkoxy (C 2 -C 4 )alkyl.
  • N-terminal derivatives of compounds of the invention may be those in which the N-terminal amino group -NH 2 is instead a group -NHR 6 wherein R 6 represents
  • (C 1 -C 4 )alkyr' represents straight or branched alkyl chains of from 1 to 4 carbon atoms, such as: methyl, ethyl, propyl, 1-methylethyl, butyl, 1-methylpropyl or 1,1-dimethylethyl while the term “(C 2 -C 4 )alkyl” represents straight or branched alkyl chains of from 2 to 4 carbon atoms such as: ethyl, propyl, 1-methylethyl, butyl, 1-methylpropyl or 1 ,1-dimethylethyl.
  • (C 5 -C 7 )cycloalkyl represents a cycloalkyl group selected from cyclopentyl, cyclohexyl and cycloheptyl.
  • (C 1 -C 4 )alkoxy represents a straight or branched alkoxy chain of 1 to 4 carbon atoms such as methoxy, ethoxy, propoxy, 1-methylethoxy, butoxy, 1-methylpropoxy and 1 ,1- dimethylethoxy.
  • Derivatives according to the present invention may be made according to the methods described for the manufacture of derivatives of actagardine in EP-0195359, the disclosure of which is incorporated herein by reference.
  • the derivative is a compound where the C-terminal is of the formula -COR, in which R represents the group -NR 1 R 2 , in some embodiments, R 1 is H and R 2 represents a group of formula -(CH 2 ) n -NR 3 R 4 , in which n represents an integer from 2 to 8 and R 3 and R 4 independently represent hydrogen or (C 1 -C 4 ) alkyl or R 3 and R 4 taken together represent a group -(CHa) 3 -, -(CH 2 J 4 -, (CH 2 )Z-O-(CHz) 2 -, -(CH 2 )2-S-(CH 2 ) 2 - or -(CHz) 5 -.
  • R 3 and R 4 preferably represent hydrogen or (C 1 -C 4 ) alkyl. More preferably R 3 and R 4 represent (C 1 -C 2 ) alkyl, e.g. methyl.
  • Integer n may be preferably from 2 to 5, and more preferably 2 to 4, e.g. 3.
  • R 1 and R 2 taken together with the adjacent nitrogen atom represent a piperazine moiety.
  • the N-substituent in the 4 position may preferably be selected from:
  • the piperdinyl and substituted piperidinyl groups preferably have their nitrogen atom at the 4- position.
  • the N-substituent may more preferably be selected from:
  • R 5 and R 6 may be preferably (C r C 4 )alkyl, more preferably (C 1 -C 2 ) alkyl, e.g. methyl.
  • Integer p is preferably 1 to 4, e.g. 3.
  • the N substituent is substituted piperidinyl, then the N-substituent is preferably (CVC 2 ) alkyl, e.g. methyl.
  • the N is preferably in the 4-position.
  • a nucleic acid of the invention may be a DNA or RNA, though preferably a DNA.
  • a nucleic acid of the invention may be single- or double-stranded.
  • the invention provides an isolated nucleic acid encoding a cluster polypeptide.
  • the invention provides an isolated nucleic acid encoding a precursor polypeptide or variant or fragment thereof.
  • the invention provides an isolated nucleic acid which may comprise all or a fragment of SEQ ID NO:100 or SEQ ID NO:200, including a fragment comprising an intergenic region disclosed herein.
  • Such regions may include a promoter or other regulatory element for the expression of a cluster polypeptide or a precursor polypeptide of the present invention.
  • fragments include fragments of SEQ ID NO:100 or SEQ ID NO:200, or variants thereof having significant sequence identity, which are at least 25, e.g. at least 30, e.g. at least 50, e.g. at least 100, e.g. at least 250 nucleotides in length.
  • Promoters that are variants of those intergenic sequences are also included and the specific intergenic sequences (or parts thereof) are preferred embodiments.
  • the invention in its broader sense is intended to include embodiments having variants of that specific sequence.
  • nucleic acid denotes: any nucleic acid having a sequence which is different from that of the reference nucleic acid, but which is its complement or which shows significant nucleic acid sequence identity with, or hybridization under stringent conditions to, the reference nucleic acid or its complement or a fragment of the reference nucleic acid or its complement; or any nucleic acid which encodes an amino acid sequence having significant amino acid sequence identity with the amino acid sequence encoded by the reference nucleic acid, or a fragment of that nucleic acid.
  • variant nucleic acids of the invention are further defined as follows. If a variant nucleic acid of the invention is introduced into the gene clusters identified herein, in place of the sequence of which it is a variant, and the recombinant fragment is introduced into a suitable host cell under suitable conditions for lantibiotic production (e.g. as shown in the Examples), then production of a molecule having one or more activities of a lantibiotic (especially antibiotic activity) will result. Preferably production will be regulated to occur at high cell density.
  • nucleic acid sequence identity is preferably at least 50%, more preferably 60%, 70%, 80% or 90%, still more preferably 95%, 98% or 99%.
  • Significant nucleic acid sequence identity is preferably shown between the variant nucleic acid (or a portion thereof) and a fragment of at least 30 residues of the reference nucleic acid, more preferably a fragment of at least 60, 90 or 120 residues, still more preferably a fragment of 180, 240 or 300 residues, more preferably the entire reference nucleic acid.
  • Percent (%) nucleic acid sequence identity is defined as the percentage of nucleotide residues in a candidate sequence that are identical with the nucleotide residues in the sequence under comparison.
  • the identity values used herein were generated by the BLASTN module of WU BLAST-2 set to the default parameters, with overlap span and overlap fraction set to 1 and 0.125, respectively.
  • nucleic acid sequence identity is preferably assessed over a sequence of at least 30 residues, more preferably 40 or 50 residues, still more preferably 60 residues.
  • “Stringent conditions” or “high stringency conditions”, as defined herein, may be identified by those that: (1) employ low ionic strength and high temperature for washing, for example 0.015 M sodium chloride/0. 0015 M sodium citrate/ 0.1% sodium dodecyi sulfate at 5O 0 C; (2) employ during hybridization a denaturing agent, such as formamide, for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42°C; or (3) employ 50% formamide, 5 x SSC (0.75 M NaCI, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50 g/ml), 0.1% SDS, and 10% dex
  • nucleic acid of interest When a nucleic acid of interest is said to be in "operative association" with a promoter or regulatory sequence, this means that the promoter/regulatory sequence is able to direct transcription of the nucleic acid of interest in an appropriate expression system, with the nucleic acid of interest in the correct reading frame for translation.
  • the transcript of the nucleic acid of interest contains an appropriately located ribosome binding site for expression in an appropriate expression system of the polypeptide encoded by the nucleic acid of interest. See for example Sambrook et al. (1989) and Ausubel et al. 25 (1995).
  • nucleic acid When a nucleic acid is referred to as "isolated”, this may mean substantially or completely isolated from some or all other nucleic acid normally present in A. garbadinensis and/or A. liguriae, especially nucleic acid from outside the gene cluster segments identified herein.
  • this invention provides nucleotide sequences or a set of nucleotide sequences encoding the actagardine or actagardine B biosynthetic gene cluster. Accordingly, the entire gene cluster or portions thereof of at least twenty-five contiguous nucleotides may be used for a wide variety of applications, including but not limited to: expression of actagardine or actagardine B; use as probes to screen other organisms for related molecules and the like; use to induce gene silencing and the like.
  • an expression construct comprising a nucleic acid encoding a cluster polypeptide or a lantibiotic polypeptide of the invention operably linked to a promoter.
  • a set of expression constructs comprises two or more polypeptide coding sequences of the present invention and at least one promoter suitable for the expression of said sequences.
  • the promoter(s) may be a promoter with which the polypeptide gene is naturally associated with (or in the case of a variant, the promoter of the gene from which the variant is derived), or may be a constitutive or inducible promoter functional in the host cell. Promoters thus include intergenic regions of SEQ ID NO:100 or SEQ ID NO:200 upstream of any of the open reading frames listed in Tables 1 and 2.
  • the promoter(s) will be operably linked to the nucleic acids of the set of expression constructs.
  • operably linked it will be understood that the promoter will be able to direct transcription of the nucleic acid of interest in an appropriate expression system, with the nucleic acid of interest in the correct reading frame for translation.
  • the transcript of the nucleic acid of interest contains an appropriately located ribosome binding site for expression in an appropriate expression system of the polypeptide encoded by the nucleic acid of interest. See for example Sambrook et al. (1989), Ausubel et al. (2002) and Kieser (2000).
  • Sets of expression constructs according to the invention include numerous permutations of genes encoding precursor and cluster polypeptides of the invention as defined above.
  • the set will include at least a LanA gene. Examples of such sets are set out as “Set 1" to “Set 7" below, though these sets should be understood to be merely illustrative and not limiting.
  • a LanA gene encoding a precursor polypeptide, preferably a precursor polypeptide capable of being converted to a compound of the invention, plus a LanM gene encoding a LanM polypeptide.
  • the LanM polypeptide is preferably a LanM of SEQ ID NO:120 or a variant thereof, or SEQ ID NO:213 or a variant thereof.
  • a LanA gene encoding a precursor polypeptide, preferably a precursor polypeptide capable of being converted to a compound of the invention, plus a LanR gene encoding a LanR polypeptide.
  • the LanR polypeptide is preferably a LanR of SEQ ID NO:122 or a variant thereof, or SEQ ID NO:216 or a variant thereof.
  • a LanA gene encoding a precursor polypeptide, preferably a precursor polypeptide capable of being converted to a compound of the invention, plus a LanM gene encoding a LanM polypeptide, plus a LanR gene endcoding a LanR polypeptide.
  • the LanM polypeptide is preferably a LanM of SEQ ID NO:120 or a variant thereof, or SEQ ID NO:213 or a variant thereof.
  • the LanR polypeptide is preferably a LanR of SEQ ID NO:122 or a variant thereof, or SEQ ID NO:216 or a variant thereof.
  • the genes of Set 3 together with a LanO gene encoding a LanO polypeptide is preferably SEQ ID NO:122 or a variant thereof, or SEQ ID NO:215 or a variant thereof.
  • Set 5 The genes of Set 3 or Set 4 together with a LanT gene encoding a LanT polypeptide.
  • the LanT polypeptide is preferably SEQ ID NO:123 or a variant thereof, or SEQ ID NO:214 or a variant thereof.
  • Set 6 The genes of SEQ ID NOs:116 to 127 or variants thereof.
  • Set 7 The genes of SEQ ID Nos:206 to 220 or variants thereof.
  • a set will comprises sequences which all encode 1xx polypeptides or which all encode 2xx polypeptides. However sets which are made up of both 1xx and 2xx polypeptides are not excluded from the present invention.
  • a recombinant vector comprising one or more expression constructs of the invention.
  • a set of recombinant vectors which comprise a set of expression constructs of the invention.
  • Suitable vectors comprising nucleic acid for introduction into bacteria can be chosen or constructed, containing appropriate additional regulatory elements if necessary, including additional promoters, terminator fragments, enhancer elements, marker genes and other elements as appropriate.
  • Vectors may be plasmids, viral e.g. phage, or phagemid, as appropriate. For further details see, for example, Sambrook et al (1989) Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor.
  • the inventors have developed a vector system useful for producing and screening lantibiotic derivatives of actagardine B. This is achieved by introducing one or more restriction endonuclease recognition sites into the LanA gene which encodes SEQ ID NO:1 , 11 or 212 in order to produce an expression cassette system.
  • the invention provides a recombinant DNA cassette which comprises a nucleotide sequence encoding an actagardine B precursor polypeptide, wherein said sequence comprises a first restriction site at or adjacent the N-terminal encoding region of the encoding sequence; optionally a second restriction site downstream of the first restriction site and within the encoding sequence; and a third restriction site at or adjacent the C-terminal encoding region of the encoding sequence, wherein at least one of said restriction sites does not occur within the LanA coding sequence shown as SEQ ID NO:200.
  • a recombinant DNA cassette which comprises a nucleotide sequence encoding an actagardine precursor polypeptide, wherein said sequence comprises a first restriction site at or adjacent the N-terminal encoding region of the encoding sequence; optionally a second restriction site downstream of the first restriction site and within the encoding sequence; and a third restriction site at or adjacent the C-terminal encoding region of the encoding sequence, wherein at least one of said restriction sites does not occur within the LanA coding sequence shown as SEQ ID NO:100.
  • all two or three sites will be different from each other. It is also desirable that when the cassette is carried by a vector, the sites are unique for that vector.
  • the non-naturally occurring restriction enzyme site is the second restriction site and is located between codons 5 and 16, such as between 6 and 15, of the encoding sequence of SEQ ID NO:1 or SEQ ID NO:4.
  • the cassette will desirably also include a LanA leader sequence and a LanA promoter, and may include in addition one or more cluster genes, particularly where such a cluster gene is required to complement the loss of the equivalent host cell gene.
  • the cassette of the invention described above may be engineered in a variety of ways.
  • the fragment obtained by cleaving the cassette between the first and second, first and third, or second and third, restriction sites may be replaced with a variant coding sequence encoding a .lantibiotic A variant.
  • the invention provides a variant of the cassette of the invention wherein said variant has from 1 to 15 nucleotide substitutions within the encoding region of the encoding sequence.
  • the sequence of between the first and second, first and third, or second and third, restriction sites may be replaced by a larger stuffer fragment.
  • the cassette encoding a Iantibiotic derivative may be used to transform a host cell to express the derivative, for example to assess its anti-bacterial properties.
  • a multiplicity of expression cassettes may be made to provide a library of different derivatives, which may then be screened for activity.
  • An expression cassette of the invention may be based on any cloning and expression vector used in the art for the expression of genes in host cells.
  • Such vectors will include one or more origins of replication, which may be temperature sensitive.
  • the vectors may include a selectable marker, such as the chloramphenicol acetyl transferase gene, the erythromycin resistance gene, the apramycin resistance gene or the tetracycline resistance gene.
  • the vector may also contain a targeting region, this region being homologous to a genomic sequence present in the host cell outside the LanA gene cluster. Such a vector may be used to integrate the cassette into the genomic sequence homologous to the targeting region.
  • the expression cassette may also comprise one or more cluster genes in addition to the LanA gene or derivative thereof.
  • the host cell is a ⁇ LanA host cell in which the LanA gene has been inactivated in a manner which also inactivates such a cluster gene (e.g. in the strain disclosed in Altena et al, 2000), it is desirable that this gene or an equivalent gene is provided on the expression cassette.
  • the first base of the restriction site is located at a position from six residues upstream of the ATG codon of the LanA leader sequence to no more than six codons downstream of the first codon of the propeptide.
  • the first base of the restriction site is located at a position from twelve, preferably six, residues upstream to six residues downstream of the first codon of the propeptide encoding sequence.
  • the first restriction site is a BgIW site.
  • the first base of the restriction site either includes at least one of the nucleotides of the termination codon of the propeptide or the 5' or 3' nucleotide of the restriction site is no more than twelve, preferably six, residues downstream or upstream respectively of the termination codon.
  • the third restriction site is a Av ⁇ site.
  • the second restriction site when present, will lie between the first and third restriction sites.
  • the restriction site includes at least one nucleotide present from codon 5 to codon 16, preferably codon 8 to 16 of the propeptide-encoding sequence.
  • a SS ⁇ G1 site has been introduced by altering codons 6 and 7 of the ActA-encoding sequence.
  • other changes are also contemplated by the present invention.
  • the expression cassette includes two or more sites between the first and third restriction sites.
  • the cassette may include two or three non-naturally occurring restriction sites. In the accompanying example, all three sites do not normally occur in the ActA sequence encoded by of SEQ ID NO:100.
  • the expression cassette simplifies the rapid production of lantibiotic derivatives, as discussed further herein below.
  • the region between the first and second sites, the first and third, or the second and third sites may be replaced by a stuffer fragment.
  • a stuffer fragment is a piece of DNA which is larger than the sequence which it replaces.
  • the stuffer fragment may be from 50 to 5000 nucleotides in size, for example from about 500 to 2000 nucleotides in size. The value of introducing these stuffer DNA fragments is that when the region is replaced by a lantibiotic-encoding oligonucleotide there is a significant decrease in plasmid size. The resulting plasmid can thus be readily purified away from any minor population of unrestricted plasmid thus eliminating any background.
  • a cassette of the invention may be used to introduce specific changes to the ActA sequence in a vector which can then be introduced into a host cell for expression of a lantibiotic.
  • the sequence is desirably operably linked to the LanA (e.g. ActA or LJgA) leader sequence, which in turn is operably linked to the LanA promoter (e.g. ActA or LJgA)
  • the vector comprising the cassette may also include a LanR gene.
  • the LanR gene will be located downstream of, and in tandem with, the lantibiotic A coding sequence.
  • Expression cassettes of the invention may be used to provide libraries of lantibiotic-encoding genes. Such libraries may be made by introducing into the cassette, between the first and second restriction sites, the first and third restriction sites, or the second and third restriction sites, a multiplicity of sequences each of which corresponds to the corresponding AdA or LigA sequence apart from having from 1 to 15, for example from 1 to 10, preferably from 1 to 6, for example from 1 to 3 nucleotide changes compared to the propeptide portion of SEQ ID
  • libraries form a further aspect of the invention.
  • Such libraries may comprise from 10 to 100,000, such as from 10 to 10,000 for example from 10 to 1 ,000 different coding sequences which are variants of the lantibiotic A coding sequence of an expression cassette.
  • An expression cassette encoding a lantibiotic A derivative may be introduced into a host cell for expression of the lantibiotic.
  • the library may be transformed into host cells, and colonies isolated and/or screened for antibacterial activity.
  • the sequences of the lantibiotic A expressed by individual colonies showing such activity can be determined.
  • the invention further provides a lantibiotic obtained by the method of the invention.
  • the first type of host cell is a lantibiotic producing host cell.
  • the host cell may be a non-producer cell, i.e. does not contain a LanA gene or its associated cluster genes required for producing a LanA polypeptide.
  • the invention provides a host cell transformed with a set of expression constructs of the invention.
  • the set of constructs may be any one of Sets 1 to 7 as defined above, or a set based upon any other combination of precursor and cluster polypeptide- encoding nucleic acids.
  • the host cell may be transformed with a expression cassette of the invention.
  • the host cell may be a lantibiotic producing host cell.
  • a lantibiotic producing host cell is one in which an expression construct comprising a LanA gene, if introduced into the cell in the absence of any cluster gene, would be expressed and a LanA polypeptide produced.
  • Such cells include any type-B lantibiotic producing cell such as any strain of a bacillus, an actinomycete, or a streptomycete, (e.g. S. lividans or S. coe//co/or ⁇ which produces a lantibiotic.
  • Examples of such cells include a cinnamycin-producing host cell (Streptomyces cinnamoneus cinnamoneus DSM 40005), or an actagardine-producing Actinoplanes garbadinensis or A. liguriae NCIMB 41362.
  • the host cell may be A. liguriae NCIMB 41362 without any further modification.
  • a host cell of this class may comprise a mutation in its endogenous LantibioticA gene such that the gene is not expressed or the gene product is inactive.
  • Such a host cell may be obtained by targeted homologous recombination to delete or mutate the LanA gene of the host cell. Methods to achieve this are known as such in the art and are illustrated in Altena et al, (2000) and WO2005/093069, the disclosures of which are incorporated herein by reference.
  • the resulting host cell is referred to as a ALanA host cell.
  • the host cell is a ALigA A.
  • the host cell is a AAdA A. garbadinensis host cell in which the ActA gene has been inactivated, for example by mutation or deletion, e.g. deletion brought about by homologous recombination.
  • a non-producer cell may be any host cell in which expression of a LanA gene encoding a precursor polypeptide capable of being converted to actagardine or a variant thereof, or to a compound of the invention, can produce such a product provided the LanA gene is introduced into the cell as part of a set of expression constructs which are capable of converting a precursor polypeptide to actagardine or a variant thereof, or to a compound of the invention.
  • a non-producer host cell may be a bacterial host cell.
  • Bacterial host cells include an actinomycete, or a streptomycete, e.g. S. Hvidans ,S. coelicolor or S. cinnamoneus.
  • Compounds of the invention may be produced by expression of a nucleic acid, for example in the form of an expression construct encoding a precursor polypeptide carried in a recombinant expression vector, in a host cell which carries a LanA gene together with where necessary, associated cluster genes required for conversion of the precursor polypeptide to the product.
  • a host cell which carries a LanA gene together with where necessary, associated cluster genes required for conversion of the precursor polypeptide to the product.
  • the host cell may be A. liguriae NCIMB 41362 without any further modification.
  • the introduction of the expression cassette, or vector(s) into a host cell may (particularly for in vitro introduction) be generally referred to without limitation as "transformation".
  • transformation may employ any available technique.
  • suitable techniques may include calcium chloride transformation, polyethyleneglycol assisted transformation, electroporation, conjugation and transfection or transduction using bacteriophages.
  • the present invention provides a method of expressing nucleic acid of the invention, the method comprising providing a host cell (or other expression system) culturing the host cell, so as to express the nucleic acid of interest.
  • the nucleic acid of interest will be in an expression cassette, such that culturing the host cell leads to the production of a product of the invention.
  • the nucleic acid of interest is expressed substantially only when the host cell culture reaches high cell density, more preferably at or close to the stationary phase of host cell culture.
  • Cell cultures at or close to stationary phase may have OD650 values in the range of 1- 20.
  • Known methods of culturing cells are well known in the art, for example from Sambrook et al (1989), Ausubel et al (2002), and (in particular for Streptomyces spp.) Kieser et al (2000).
  • the expression products of the expression systems may be collected and purified. Isolation methods may comprise capture from the fermentation medium using solvent extraction techniques, adsorption resin such as hydrophobic resins or precipitation methods such as ammonium sulfate precipitation. Purification methods may include chromatography techniques such as ion exchange, hydrophobic interaction, reverse phase, normal phase, solid phase extraction and HPLC, e.g. as described in US 5,112,806 for the isolation of mersacidin
  • the compounds of the invention may be recovered from the host cell culture.
  • the recovered compounds may be formulated in the form of a pharmaceutical composition, optionally in the form of a pharmaceutically acceptable salt.
  • host cells produce a mixture of compounds of the invention, e.g. those in which Y is -S- or -S(O)- or those in which Z is NH 2 or AIa-, or mixtures of all four types
  • the products may be isolated using standard separation techniques such as hplc, e.g. as described in US 6,022,851 for the production of Actagardine and Ala-Actagardine.
  • the recovered compounds may be formulated in the form of a pharmaceutical composition, optionally in the form of a pharmaceutically acceptable salt.
  • a “pharmaceutically acceptable salt” may be an acid addition salt in which the base retains the biological effectiveness and properties of the compound and which is physiologically acceptable.
  • Such salts include those formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulphuric acid, nitric acid, phosphoric acid and the like, and organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like.
  • Salts also include basic salts, such as an alkali or alkaline earth metal salt, e.g. a sodium, potassium, calcium or magnesium salt.
  • an alkali or alkaline earth metal salt e.g. a sodium, potassium, calcium or magnesium salt.
  • the [antibiotics of the present invention may be formulated together with one or more other pharmaceutically acceptable ingredients well known to those skilled in the art, including, but not limited to, pharmaceutically acceptable carriers, adjuvants, excipients, diluents, fillers, buffers, preservatives, anti-oxidants, lubricants, stabilizers, solubilisers, surfactants (e.g., wetting agents), masking agents, colouring agents, flavouring agents, and sweetening agents.
  • the formulation may further comprise other active agents, for example, other therapeutic or prophylactic agents.
  • the present invention further provides pharmaceutical compositions, as defined above, and methods of making a pharmaceutical composition
  • a pharmaceutical composition comprising admixing at least one active compound, as defined above, together with one or more other pharmaceutically acceptable ingredients well known to those skilled in the art, e.g., carriers, adjuvants, excipients, etc. If formulated as discrete units (e.g., tablets, etc.), each unit contains a predetermined amount (dosage) of the active compound.
  • pharmaceutically acceptable refers to compounds, ingredients, materials, compositions, dosage forms, etc., which are, within the scope of sound medical judgement, suitable for use in contact with the tissues of the subject in question (e.g., human) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • Each carrier, adjuvant, excipient, etc. must also be “acceptable” in the sense of being compatible with the other ingredients of the formulation.
  • compositions may be formulated for any suitable route and means of administration.
  • Pharmaceutically acceptable carriers or diluents include those used in formulations suitable for oral, rectal, nasal, topical (including buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural) administration.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
  • conventional non-toxic solid carriers include, for example, pharmaceutical grades of mannitol, lactose, cellulose, cellulose derivatives, starch, magnesium stearate, sodium saccharin, talcum, glucose, sucrose, magnesium carbonate, and the like may be used.
  • the active compound as defined above may be formulated as suppositories using, for example, polyalkylene glycols, acetylated triglycerides and the like, as the carrier.
  • Liquid pharmaceutically administrable compositions can, for example, be prepared by dissolving, dispersing, etc, an active compound as defined above and optional pharmaceutical adjuvants in a carrier, such as, for example, water, saline aqueous dextrose, glycerol, ethanol, and the like, to thereby form a solution or suspension.
  • a carrier such as, for example, water, saline aqueous dextrose, glycerol, ethanol, and the like
  • the pharmaceutical composition to be administered may also contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like, for example, sodium acetate, sorbitan monolaurate, triethanolamine sodium acetate, sorbitan monolaurate, triethanolamine oleate, etc.
  • composition or formulation to be administered will, in any event, contain a quantity of the active compound(s) in an amount effective to alleviate the symptoms of the subject being treated.
  • Dosage forms or compositions containing active ingredient in the range of 0.25 to 95% with the balance made up from non-toxic carrier may be prepared.
  • a pharmaceutically acceptable non-toxic composition is formed by the incorporation of any of the normally employed excipients, such as, for example, pharmaceutical grades of mannitol, lactose, cellulose, cellulose derivatives, sodium crosscarmellose, starch, magnesium stearate, sodium saccharin, talcum, glucose, sucrose, magnesium, carbonate, and the like.
  • excipients such as, for example, pharmaceutical grades of mannitol, lactose, cellulose, cellulose derivatives, sodium crosscarmellose, starch, magnesium stearate, sodium saccharin, talcum, glucose, sucrose, magnesium, carbonate, and the like.
  • Such compositions take the form of solutions, suspensions, tablets, pills, capsules, powders, sustained release formulations and the like.
  • Such compositions may contain 1%-95% active ingredient, more preferably 2-50%, most preferably 5-8%.
  • Parenteral administration is generally characterized by injection, either subcutaneously, intramuscularly or intravenously.
  • Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions.
  • Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol or the like.
  • the pharmaceutical compositions to be administered may also contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like, such as for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate, triethanolamine sodium acetate, etc.
  • the pharmaceutically acceptable compositions may be formulated in a suitable ointment or gel containing the active component suspended or dissolved in one or more carriers.
  • Carriers for topical administration of the compounds of this invention include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water.
  • the pharmaceutically acceptable compositions can be formulated in a suitable lotion or cream containing the active components suspended or dissolved in one or more pharmaceutically acceptable carriers.
  • Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.
  • the percentage of active compound contained in such parental or topical compositions is highly dependent on the specific nature thereof, as well as the activity of the compound and the needs of the subject. However, percentages of active ingredient of 0.1% to 10% w/w employable, and will be higher if the composition is a solid which will be subsequently diluted to the above percentages.
  • the composition will comprise 0.2-2% w/w of the active agent in solution.
  • Administration of Compounds Lantibiotic compounds and compositions of the invention may be administered to a subject in a method of medical treatment or prophylaxis.
  • the subject may be a human or animal subject.
  • the animal subject may be a mammal, or other vertebrate.
  • a compound of the invention for use in a method of treatment or prophylaxis of a subject.
  • a compound of the invention for the manufacture of a medicament for use in a method of treatment or prophylaxis of a subject.
  • the method of treatment may be of a bacterial infection, including a skin, mucosal, enteric or systemic infection.
  • the variants and composition may be used for systemic treatment of bacteraemia (including catheter related bacteraemia), pneumonia, skin and skin structure infections (including surgical site infections), endocarditis and osteomyelitis. These and other such treatments may be directed against causative agents such as staphylococci, streptococci, enterococci.
  • the compounds of the invention or compositions thereof may also be used for topical treatment of skin infections including acne ie. Propionibacterium acnes.
  • the variants and compositions thereof may also be used in the treatment of eye infections, such as conjunctivitis, and for oral treatment for gut super-infection, such as that caused by Clostridium difficile including multiply-resistant C. difficile (pseudomembranous colitis), or gut infections associated with Helicobacter pylori.
  • the variants may also be used in the treatment or prevention of infection of the skin in wounds or burns.
  • the variants and compositions thereof may be used in prophylactic methods, such as for the clearance of the nares to prevent transmission of MRSA. This may be practiced on subjects at risk of infection (e.g. patients entering a hospital) or on health professionals or other carers at risk of being carriers of such infections. Prophylactic clearance of gut flora ahead of abdominal surgery is also contemplated.
  • the compounds according to the invention can be administered enterally (orally), parenterally (intramuscularly or intravenously), rectally or locally (topically). They can be administered in the form of solutions, powders (tablets, capsules including microcapsules), ointments (creams or gel), or suppositories. Possible auxiliaries for formulations of this type are the pharmaceutically customary liquid or solid fillers and extenders, solvents, emulsifiers, lubricants, flavor corrigents, colorants and/or buffer substances. As an expedient dose, 0.1- 1000, preferably 0.2-100, mg/kg of body weight are administered. They are expediently administered in dose units which contain at least the efficacious daily amount of the compounds according to the invention, e.g. 30-3000, preferably 50-1000, mg.
  • O/SBDIG-1 is a digoxigenin (DIG)-labelled degenerate oligonucleotide composed of 48 bases. It was designed by translating the known amino acid sequence of actagardine and considering codon usage for Actinoplanes. Southern hybridisation analysis of genomic DNA isolated from A. garbadinensis and digested using the restriction enzyme Nco ⁇ , identified a ⁇ 3kb fragment which hybridised to O/SBDIG-1. The Nco ⁇ digest of the genomic DNA was repeated and DNA fragments of ⁇ 3kb were isolated and cloned into Nco ⁇ cut pLITMUS28 (NEB). The resulting plasmids were introduced into E.
  • DIG digoxigenin
  • pLITAGOI consists of DNA encoding the lanA structural gene for actagardine biosynthesis ⁇ actA) together with an upstream region believed to encode a portion of an ABC sugar transporter and a region downstream partially encoding Ian M (actM).
  • the primers O/ACT08F and O/ACT09R were designed based upon sequence from pLITAGOI . Using these primers in a PCR reaction together with DIG-labelled dNTPs (Roche) and pLITAGOI as a template, a 2296bp DIG-labelled DNA fragment was generated and designated SBDIG-2.
  • cosmid libraries Two cosmid libraries were generated by cloning Sau3A ⁇ digested genomic DNA from A. garbadinensis ATCC 31049 and A. liguriae NCIMB 41362, into the cosmid SuperCosi (Stratagene) previously digested using BamH ⁇ . Each cosmid library was analysed by Southern hybridisation using SBDIG-1. Twenty-five cosmids from each library believed to hybridise to SBDIG-1 were selected and re-analysed via Southern hybridisation using the probes O/SBDIG-1 and SBDIG-2. Nine cosmids derived from genomic DNA from A. garbadinensis and eleven cosmids derived from genomic DNA from A. liguriae hybridised to both probes. DNA was isolated from each cosmid and sequenced using the primers T3 and 17. The cosmids CosAL02 and CosAG14 were subsequently fully sequenced (Sequencing facility, Department of Biochemistry, University of Cambridge).
  • Bacterial strains used in the present invention are summarised in Table 5.
  • the cosmid SuperCosi was obtained from Stratagene.
  • the plasmid pLITMUS was obtained from New England Biolabs. Primers
  • DNA hybridisation probes were prepared using the Digoxygenin (DIG) PCR DNA labeling and detection kit supplied by Roche, according to their instructions.
  • DIG Digoxygenin
  • the DNA of interest was initially separated by agarose gel electrophoresis and transferred to a nylon membrane (Hybond-N, Amersham Int., UK) using a vacuum blotter (Q BIO gene).
  • the time taken for depurination of the DNA using 0.5 M HCI was judged by the time taken for the bromophenol blue marker band to turn completely yellow (typically 15-20 min for a 0.7% agarose gel).
  • the DNA was then systematically denatured with 1.5 M NaCI, 1.5 M NaOH and then neutralised using 0.5 M Tris, 1.5 M NaCI, pH 8.0 for a further 15-20 min each. Complete blotting of the DNA was facilitated by flooding with 20 x SSC for a minimum of 60 min.
  • the DNA was cross-linked by placing the membrane (DNA face down) on a UV transilluminator (UVP) and exposing it to UV at a wavelength of 365 nm for 5 min.
  • UVP UV transilluminator
  • Colonies to be screened by hybridisation were transferred onto a nylon membrane (Roche diagnostics). This was achieved by placing the positively charged nylon membrane over the colonies and pressing firmly for 1 min to ensure effective transfer. Reference points were marked on the membrane to indicate its orientation with respect to the colonies. Following this, the membrane was removed and prepared for hybridisation as directed in the Roche user's manual (DIG Application manual for filter hybridisation).
  • the cosmid, CosAG14 contains a 38168bp fragment of genomic DNA isolated from A. garbadinensis. Sequence analysis has identifed DNA encoding the leader and actagardine prepeptide, this gene has been assigned the name actA. Two alanine residues lie immediately upstream of the actagardine prepeptide. These residues are believed to represent the recognition site for protease cleavage of the leader peptide from actagardine. Partial cleavage at this position resulting in the retention of an alanine is thought to result in the production of ala-actagardine routinely observed in fermentation broths of A. garbadinensis.
  • actA Downstream of the actA gene lies a 3162bp region of DNA with strong sequence similarity to several lanM proteins, for example, mrsM (30% identity) from the mersacidin gene cluster.
  • actM This putative gene has been designated actM and is thought to be involved in the modification of the actagardine prepeptide, catalysing dehydration and thioether formation.
  • actO An open reading frame designated actO, that is located 11bp downstream of actM encodes a 341-amino-acid protein with sequence similarity ( ⁇ 39% identity) to several luciferase-type monooxygenases.
  • actO The role of the monooxygenase, ActO, is believed to be to catalyse the incorporation of oxygen generating actagardine from deoxy-actagardine.
  • actR In reverse orientation to actO and located 62bp downstream is the open reading frame named actR.
  • the protein product of this orf shows sequence similarity (-37% identity) to several two-component response regulators.
  • Positioned 789bp downstream and in the same orientation to actR lies a putative 812 amino- acid protein that shows sequence similarity (-25% identity) to ABC transporter permeases. This putative protein designated actT is potentially reponsible for the export of the modified lantibiotic from the cell.
  • the cosmid CosAL02 contains a 40402bp fragment of genomic DNA isolated from Actinoplanes liguriae. Sequence analysis has identified a lanA gene encoding a 64-amino-acid protein with strong sequence homology (50 identical residues) to the actA gene identified in the cosmid CosAG14. We have termed this species of lanA gene as HgA. The amino acid sequence of the prepeptide of this lanA differs from that of actagardine by two residues indicated in the alignment of the two genes shown below (SEQ. ID 119 and SEQ. ID 212):
  • the mutations V15L and 116V would generate a protein with an identical mass to actagardine and would therefore not be distinguished by mass spectroscopy (lc-ms) analysis.
  • the potential product of the lanA gene identified in CosAL02 represents a novel lantibiotic.
  • An open reading frame that lies 321 bp upstream of HgA encodes a putative 286-amino acid protein that shows sequence similarity (46% identity) to the StrR protein of Streptomyces glaucescens.
  • the StrR protein is a pathway-specific DNA binding activator involved in the regulation of streptomycin gene expression.
  • HgM 1046-amino-acid lanM polypeptide
  • CosAG14 the next orf downstream of HgT encodes for a 347 amino-acid protein with sequence similarity (-38% identity) to luciferase-type monooxygenases.
  • This putative monooxygenase (MgO) is believed to be involved in the incorporation of oxygen and sulfoxide bond formation.
  • ligR Positioned downstream of HgO and in reverse orientation lies a putative 217 amino-acid protein that shows sequence similarity (-37%) to several two-component response regulators. This putative regulator has been designated ligR.
  • Table 1 Annotation of CosAG14 (38168bp fragment isolated from A. garbadinensis. The SuperCosi vector backbone sequence is omitted)
  • This example illustrates the production of an expression cassette according to the present invention.
  • This expression cassette, plasmid pAGvarX has been designed for the efficient generation of variant lanA genes of the present invention which can then be introduced into a host cell, such as a strain of A. garbadinensis in which the wild-type actA has been removed (A. garbadinensis A actA).
  • This plasmid a derivative of the vector pSET152 (Bierman et al., 1992) will integrate into the host's chromosome via the attP attachment site. Expression of the mutated actA gene by the host organism together with the remaining wild-type genes of the actagardine biosynthetic gene cluster should generate actagardine variants. Construction of the plasmid pAGvarX
  • the region of DNA spanning from the C-terminus of the actA to the adjacent downstream orf (21758-21836 inclusive) was amplified by PCR using the primers G7AGvarO5F and O/AGvar06R and pLITAGOI as a template.
  • the primers were designed to introduce a flanking Av ⁇ site at the 5' position and an EcoRI site at the 3' end.
  • the resulting PCR product was cloned into dephosphorylated pUC19 previously digested using Smal to yield pAGvar2.
  • the plasmids pAGvari and pAGvar2 were then digested using Xba ⁇ and the PCR fragment from pAGvari recovered and cloned into dephosphorylated Xba ⁇ digested pAGvar2, the correct orientation of the incoming fragment was determined by restriction analysis.
  • the resulting plasmid pAGvar3 was subsequently digested using BgIW and Av ⁇ and ligated to the annealed oligonucleotides O/AGvar03F and O/AGvar04R generating pAGvar4.
  • the plasmid pAGvar4 was subsequently digested using EcoRI and Xba ⁇ and the resulting ⁇ 620bp fragment including the annealed oligonucleotides introduced into pSET152 previously digested using EcoRI and Xba ⁇ yielding the vector pAGvarX.
  • the region of pAGvarX constructed by annealing the respective oligonucleotides introduce a BsrG ⁇ site via a silent mutation at the amino acids 6 and 7 (C and T respectively) with respect to the actagardine peptide.
  • This site can be used in conjunction with either the upstream BgIW site or downstream Av ⁇ site to introduce DNA encoding targeted mutations to any of the amino acids encoded within the actA peptide.
  • This example illustrates the production of a lantibiotic-producing host cell in which the lanA gene has been inactivated.
  • the host cell is A. garbadinensis in which the actA gene has been deleted. Construction of the strain A. parbadinensis ⁇ actA
  • the strain A. garbadinensis ⁇ actA is utilized as a host for expressing variants of the actagardine structural gene actA.
  • This strain was generated from wild-type A. garbadinensis using the Redirect technology developed by Gust et a/., 2002. Firstly, the region of DNA from the cosmid CosAG14 encoding actA was replaced with the cassette SBdel-1.
  • SBdel-1 consists of the apramycin resistance gene (aac(3)IV) and or/7 flanked by FLP recognition target (FRT) sites and was amplified by PCR using the plasmid plJ773 as the template together with the primers O/SB50F and O/SB51R which bind at 21536 and 21802 of SEQ ID NO:100 respectively.
  • actA of CosAG14 was replaced with SBdel-1 generating the cosmid CosAG14 ⁇ A.
  • the central part of the SBdel- 1 cassette was subsequently removed from CosAG14 ⁇ A by FLP-mediated excision following step 7 of the Redirect protocol generating CosAG14 ⁇ B. Removal of this region allows the generation of non-polar, unmarked in-frame deletions as well as repeated use of the same resistance marker (Gust et al., 2003).
  • the second stage of construction was to engineer the cosmid so that it could be introduced into A. garbadinensis via conjugation. This began by first inserting CosAG14 ⁇ B into the E. coli strain BW25113/plJ790 by transformation. The ampicillin gene of CosAG14 ⁇ B was then replaced with SBdel-2 following the Redirect protocol (Gust eif al., 2004) generating the cosmid CosAG14 ⁇ C.
  • the cassette SBdel-2 like SBdel-1 , houses the apramycin resistance gene (aac(3)IV) and oriT flanked by FRT sites but was generated using the primers O/SB52F and O/SB53R together with the template plJ773.
  • CosAG14 ⁇ C was used to tranform electrocompetent cells of E. coli ET12567/pUZ8002 before being conjugated with A. garbadinensis following the Redirect protocol (Gust et al., 2004; see also following paragraph).
  • the resulting strain in which the actA gene has been removed from the chromosome of the wild-type producer is A. garbadinensis A actA.
  • CosAG14 ⁇ C was used to transform electrocompetent cells of E. coli ET12567/pUZ8002 before being conjugated with A. garbadinensis.
  • Apramycin resistant exconjugants were obtained and sub-cultured through six successive rounds of growth in TSB without apramycin.
  • Cells from culture 6 were plated onto medium 65 and incubated at 30 0 C. After 5 days colonies were transferred and patched out over an area of approximately 1 cm 2 onto medium 65. After 3 days incubation at 30 0 C the patched cells were transferred to medium 65 containing apramycin at a final concentration of 50 ⁇ g/ml.
  • This example illustrates the expression of actagardine from the SEQ ID NO:100 gene cluster in a host cell which is a non-producer cell, S. lividans.
  • Such host cells provide an alternative means of generating active variants of these two peptides.
  • the cosmids CosAG14 and CosAL02 containing the biosynthetic gene clusters encoding the production of actagardine and deoxy-actagardine B do not possess an origin of transfer ⁇ oriT) necessary to facilitate conjugal transfer to a heterologous host.
  • an oriT together with a phage attachment site attP and integrase (int) can be introduced into the SuperCosi backbone of CosAG14 and CosAL02 replacing the neomycin resistance gene, neo.
  • the cosmid pMJCOSI (supplied by the JIC, Norwich) is a derivative of SuperCosi (Stratagene) in which the gene encoding for neomycin resistance has been replaced by a cassette (HEapra) which includes DNA encoding an oriT, attP, integrase (int) and apramycin resistance gene (aac(3)IV).
  • the cassette HEapra was isolated by digesting pMJCOSI with Ssp ⁇ and recovering the DNA from an agarose gel. This cassette together with CosAG14 and CosAL02 were used to generate the cosmids CosAG14HEapra and CosAL02HEapra respectively following the Redirect protocol as described by Gust et al., 2004.
  • the cosmid CosAG14HEapra was subsequently introduced into S. lividans via conjugation.
  • Apramycin resistant exconjugants of S. //V/dans/CosAG14HEapra were isolated.
  • Three exconjugants were used to inoculate TSB seed media.
  • S. lividans, A. garbadinensis and A. liguriae were grown in parallel to provide controls. Following 48 h incubation the seed cultures were used to inoculate a range of four different production media namely, AAS1 , GM1 , GM3 and TSB.
  • HPLC-MS analysis of the supernatants from the fermentations of S. //V/da ⁇ s/CosAG14HEapra confirm the presence of peaks with retention times and masses corresponding to ala(O)actagardine. These same peaks were absent from supernatants of S. lividans only.
  • Table 3 summarises the HPLC-MS analyses of supernatants from fermentation of S. lividans, S. //V/c/ans/CosAGMHEapra, A. garbadinensis and A. liguriae following incubation for 5 days.
  • MICs Minimum inhibitory concentrations for all organisms with the exception of Streptococcus pneumoniae were determined by two-fold serial antibiotic dilutions in Mueller-Hinton broth (MHB) supplemented with calcium chloride dehydrate to a final calcium concentration of 400 ⁇ g/ml.
  • Antimicrobial agent stock solutions were prepared and stored according to NCCLS standard M7-A6.
  • Actively growing broth cultures were diluted to contain 105 to 106 CFU/ml by adjusting to an absorbance of 0.2 - 0.3 at 600nm, equivalent to the McFarland 0.5 standard. They were then diluted a further 1 :100 in broth.
  • the assays were performed in duplicate in sterile 96-well microtitre plates in a total volume of 200 ⁇ l (160 ⁇ l broth, 20 ⁇ l antibiotic, 20 ⁇ l inoculum) in a concentration range from 64 ⁇ g/ml to 0.06 ⁇ g/ml.
  • the 12th well of the microtitre plate contained no antimicrobial agent. Vancomycin was used as a reference antibiotic for quality control. Plates were incubated aerobically, shaking, for 18 - 20 hours at 37°C with the MIC defined as the lowest concentration of drug that produced no visible growth.
  • MICs Minimum inhibitory concentrations
  • M7-A6, 2003 Minimum inhibitory concentrations
  • MICs were determined by two-fold serial antibiotic dilutions in Mueller-Hinton broth (MHB) or Brain Heart Infusion (BHI) broth (S. pneumoniae). Actively growing broth cultures were adjusted in sterile broth or by direct colony suspension (S. pneumoniae) to a turbidity equivalent to the McFarland 0.5 standard (1 x 10 8 CFU/ml), then further diluted in sterile broth for a final inoculum in sterile 96-well microtitre plates of approximately 5 x 10 5 CFU/ml.
  • MICs Minimum inhibitory concentrations for C. difficile were determined and antimicrobial agent stock solutions were prepared and stored according to the NCCLS reference agar dilution method for anaerobic bacteria (M11-A5, 2001). Two-fold serial antibiotic dilutions were prepared in Wilkens-Chalgren agar (WCA). Test organisms were selected from 48 hour growth on Braziers (C.C.E.Y.) agar, subcultured in Schaedler broth to a density equivalent to a McFarland 0.5 standard (1 x 10 8 CFU/ml), with a final inoculum onto WCA plates of approximately 10 5 CFU/spot.
  • Bacteroides fragilis ATCC 25285 was included as a reference control strain and Metronidazole was used as a reference antibiotic for quality control. All manipulations were performed in duplicate in ambient atmosphere in pre-reduced media with only brief exposure to oxygen. Plates were incubated anaerobically for 48 hours at 37 0 C with the MIC defined as the concentration of drug where a marked reduction occurred in the appearance of growth on the test plate compared to growth on the anaerobic control plate.
  • Propionibacterium acnes Test organisms were selected from 3-7 day growth on Wilkens-Chalgren agar (WCA) supplemented with furazolidone (1-2 ⁇ g/ml).
  • WAA Wilkens-Chalgren agar
  • furazolidone 1-2 ⁇ g/ml
  • Fresh Wilkens-Chalgren broth (WCB) was inoculated by direct colony suspension with single colonies of P. acnes and adjusted to a density equivalent to the McFarland 0.5 standard (1 x 10 8 CFU/ml), then further diluted in sterile WCB for a final inoculum in sterile 96-well microtitre plates of approximately 10 5 CFU/ml.
  • Two-fold serial antibiotic dilutions were performed in sterile water with stock solutions prepared and stored according to NCCLS standards (M11-A5, 2001).
  • the assays were performed in duplicate with Vancomycin and Clindamycin used as reference antibiotics for quality control. Plates were incubated anaerobically for 48-72 hours at 37 0 C with the MIC defined as the concentration of drug where a marked reduction occurred in the appearance of growth on the test plate compared to growth on the anaerobic control plate. All manipulations were performed in duplicate in ambient atmosphere in pre-reduced media with only brief exposure to oxygen.
  • Table 8 MIC values against mupirocin-resistant Staphylococcus . aureus
  • Micrococcus Iuteus was inoculated from frozen stock into 10ml Mueller-Hinton broth and grown overnight at 30 0 C with shaking at 200 rpm. 1 ml of this culture was used to inoculate 300 ml of Mueller-Hinton agar which was then poured into petri dishes. Wells (6 mm diameter) placed equidistant apart were made using a cork-borer and subsequently loaded with 50 ⁇ l of the respective sample. The bioassay plate was placed into a laminar air flow until the loaded samples had diffused, at which point the plates were transferred to a 30 0 C incubator and left overnight.
  • Digestions of DNA with restriction enzymes were carried out in the supplied buffers and in accordance with the manufacturer's guidelines. Typically, for preparative digests 5 ⁇ g of DNA was digested with 12 units of enzyme for 3 h at the recommended temperature. For analytical digests, 0.5 ⁇ g of DNA was digested with 2 units of enzyme for 2-3 h again at the recommended temperature. The digested DNA was analysed by agarose gel electrophoresis. Sub-culturinp exconjugants
  • Agar plugs of patched exconjugants were used to inoculate 50 ml flasks containing 8 ml TSB and 2 glass beads. The cultures were incubated at 30 0 C, 250 rpm for 10 days then 100 ⁇ l were removed and added to 10 ml TSB in a 50 ml flask containing 2 glass beads. The flasks were incubated for 2 days then 1 ml was removed and used to inoculate a 50 mi flask containing 10 ml TSB. Using 1 ml inoculum a total of six successive rounds of growth were carried out each incubated for 2 days at 3O 0 C, 250 rpm.
  • Cells from the sixth round of sub- culturing were pelleted by centrifuging at 4000 rpm for 20 minutes (Heraeus Sepatech Megafuge) then sonicated (MSE Sanyo Soniprep 150, amplitude 10-15 microns) for 30 seconds in TSB to disrupt the mycelium.
  • Serial dilutions (10 "1 to 10 "5 in TSB) of the sonicated cells were plated onto medium 65 and incubated at 30 0 C.
  • Fermentation for heterologous expression 50 ml conical flasks each containing 2 glass beads and either 8 ml TSB or AAS1 media supplemented with nalidixic acid and the appropriate selective antibiotic were inoculated using agar plugs or 250 ⁇ l of a -80 0 C glycerol stock. Following 2 - 4 days incubation at 30°C, 200 rpm, 1.2 ml (3%) per seed culture was used to inoculate 40 ml of the respective production media in 250 ml conical flasks containing 2 glass beads. These cultures were incubated at 30 0 C, 200 rpm for 9 days.1.5 ml whole broth aliquots were removed periodically from each culture for analysis by bioassay and/or HPLC-MS analysis.
  • Diaion HP-20 resin 50 g/L was added and mixed with supernatant isolated from a fermentation of A. liguriae and left overnight at 4°C. The suspension was aliquoted into Bond Elut columns (60 ml) and the resin washed sequentially with four bed volumes of water followed by three bed volumes of 25, 50, 75 and 100% methanol. HPLC analysis confirmed the presence of Deoxy-actagardine B in the 50, 75 and 100% methanol fractions. These fractions were combined then concentrated to approximately a quarter of the volume of the starting pool. The concentrate from 1L of broth was loaded onto two C18 Bond Elut columns (5 g) that had been pre-conditioned by washing with two column volumes of 100% methanol followed by two column volumes of water.
  • the columns were eluted sequentially with two column volumes of 50, 60, 70, 80, 90% methanol followed by two column volumes of 100% methanol.
  • HPLC analysis confirmed the presence of Deoxy-actagardine B in the 80, 90 and 100% methanol fractions, these fractions were pooled and concentrated to a third of the starting volume.
  • An equal volume of 40 imM potassium phosphate pH 2.5 in 50% methanol was added and the concentrate then loaded evenly onto three pre-equilibrated SCX Bond Elut columns (1g).
  • the SCX columns were initially washed with 40 mM potassium phosphate pH 2.5 in 50% methanol and then eluted using 1.5 column volumes of 250 mM potassium phosphate pH 7.0 in 50% methanol.
  • the eluent was desalted by loading onto a C18 Bond Elut column (5 g) that had been pre-conditioned with two column volumes of methanol followed by two column volumes of water. The column was washed with two column volumes of 50% and then 60% methanol. Deoxy-actagardine B was eluted following the addition of two column volumes each of 70, 80, 90 and 100% methanol. Fractions containing purified Deoxy- actagardine B as confirmed by HPLC and LC-MS analyses were pooled and evaporated to dryness.
  • Diaion HP-20 resin (50 g/L) was mixed with supernatant from a four litre fermentation of A. liguriae and left overnight at 4 0 C. The suspension was collected into a glass sinter funnel and the resin was washed sequentially with four bed volumes of water followed by four bed volumes of 50% Methanol. Deoxy-actagardineB and Ala(0)-deoxyactagardine B were eluted from the resin by washing with five bed volumes of 100% Methanol. The 100% Methanol fraction was concentrated to a third of the original volume and was then diluted by addition of water to a final concentration of 60% Methanol. The resulting solution was loaded onto four 10g C18 Bond Elut columns prior to washing with two column volumes of 50% Methanol.
  • Deoxy-actagardine B-related components were eluted from the column using two column volumes of Methanol/0.5% Formic Acid. The resulting eluent was concentrated by evaporation to 40 ml and Ala(0)-deoxy-actagardine B was separated from Deoxy-actagardine B by preparative HPLC using the conditions described in the table below.
  • Actagardine and Ala(0)-actagardine were purified using the method described for the purification of Deoxy-actagardine B from A. liguriae with the exception that preparative HPLC was required to resolve Ala(0)actagardine and Actagardine following the SCX Bond Elut step.
  • Electrophoresis of DNA was carried out as described by Sambrook eif a/., 1989.
  • Agarose gels (0.7-1%) were prepared in TAE buffer containing a final concentration of 0.1 ⁇ g/ml ethidium bromide to allow visualisation of the DNA by UV light.
  • 0.1 volumes of 10 x agarose gel loading solution was mixed with the samples.
  • DNA was excised from agarose gels and recovered using a Qiaquick gel extraction kit (Qiagen) and eluted in either sterile reverse osmosis purified water, Tris-HCI (10 mM, pH 8.5) or TE buffer.
  • DNA ligations were performed as described by Sambrook et ai, (1989) using 1 unit (U) of T4 DNA ligase in a total volume of 15 ⁇ l and incubating for 12-16 h at 16°C.
  • Viable cells were stored as glycerol suspensions by freezing 0.5 ml of the respective culture at -80 0 C with glycerol at a final concentration of 10%.
  • Single colonies of A. garbadinensis and A. liguriae were obtained by streaking 50 ⁇ l from a fermentation broth or glycerol stock onto either medium 65 or ABB13 plates.
  • PCRs Polymerase chain reactions (PCRs) were performed on a Stratagene Robocycler Gradient96.
  • 100-200 ng template DNA was mixed with 20 pmol of each oligonucleotide primer and dNTP's at 250 ⁇ M each.
  • Thermophilic DNA polymerase buffer as supplied by the manufacturer and DMSO made up 10% (v/v) each of a final volume of 50 or 100 ⁇ l reaction mixture.
  • a typical reaction began with an initial cycle of 1 min denaturation (94 0 C), 1 min, Y°C (annealing) and 30 seconds - 3 min extension (72°C), at which point 5 units of thermophilic DNA polymerase was added.
  • Plasmid DNA was prepared on a small scale (less than 20 ⁇ g preparation) by inoculating 3 ml of sterile 2TY or LB containing the appropriate antibiotic with single colonies picked from 2TY (or LA) agar plates. The cultures were incubated overnight (12-16 h) at 37 0 C and 250 rpm. The cells were collected by centrifugation at 12,000 xg for 1 min and plasmid DNA obtained using Wizard (Promega) Miniprep kits according to the manufacturer's guidelines. In the case of larger preparations of up to 100 ⁇ g of plasmid DNA, 30 ml of 2TY cultures were grown and plasmid DNA extracted using a Qiagen Midi-prep kit, following the manufacturer's instructions. All plasmid preparations were checked by a combination of restriction analysis and/or sequence analysis.
  • Cosmid DNA was prepared by inoculating 50 ml of sterile 2TY or LB containing the appropriate antibiotic with single colonies picked from 2TY (or LA) agar plates. The cultures were incubated overnight (12-16 h) at 37 0 C and 250 rpm. The cells were collected by centrifugation at 4,000rpm (Heraeus sepatech Megafuge 2.0R) for 20 min and Cosmid DNA isolated using a Qiagen Midi-prep kit according to the manufacturer's guidelines. Preparation and transformation of electrocompetent E. coli cells. Electrocompetent E. coli DH1 OB were prepared by the method of Dower et al.. (1988).
  • Genomic DNA templates were prepared using the procedure described by Kieser et al. (2000).
  • Antibiotic stock solutions were prepared in water (unless stated otherwise) and filter sterilised by passing through a 0.22 ⁇ m Millipore filter. Solutions dissolved in ethanol were not sterilised (Sambrook et al., 1989). All antibiotics were stored at -20 0 C. In media where apramycin was used, MgCI 2 was added to a final concentration of 10 mM (from a stock of 2.5 M).
  • Mobile phase A 10% acetonitrile, 0.1% formic acid 90% water.
  • B 90% acetonitrile, 0.1% formic acid, 90% water.
  • NCIMB 41362 was deposited under the Budapest Treaty on 7 December 2005 at NCIMB Ltd, Aberdeen, AB21 9YA, Scotland, UK, by Novacta Biosystems Limited.
  • SEQ ID NO: 121 orf21 actO monooxygena.se 341aa
  • VASTSAEDSVRAVLVNAVGATTAERIPVFAGDVVPAKKPDPA SEQ ID NO: 133 ⁇ SEQ ID NO: 199
  • SEQ ID NO: 200 CosAL02 (40402bp)
  • MDSPRLNGEDPRHTEVLAGFGAELPPIVVHRATMRVIDGAHRLSAARLRG DDRIRAVLFDGTEQEAYVLSVKANVTHGLPLSAAERTRAAERIITMHPDW SDRMIAASSGLGARTVGGLRRRRAASGESPAGLRSRAGRDSRVRPAGSTA GRLKAVDYLQDRPDASLREIARHAGVSPSTARDVRDRLHRGEDPIPATQR AAARPGNDSPPLRSLVQGLASDPSLRFSESGRDLLRWLIAHAVQDGEWKG LVDTIPAHSAQ ⁇ LAKIARHCSREWREFADILEKDAA
  • MIFTLAWSQVRSHPGRLLAIVAAVVLATGFLAATATFASTSDEGLRLTAA APLTTADIVLDADDTVHDPQWYQAAAGVRGVRSVDPQYARTVSVFGGDRR GSANVQSIPATASVRWFTVDEGTWPSAAGQVVADRRTLTDLGVGVGAHLD FRQGTAAPVPVTVVGSADLGFRPLTGSDYRFYAAASFFAGDTPPAALLTV TDRDRLAETVDAVGRALPPGATATDASAAADAAAGRFAGGNTQLVVLMLA FAAVALLASILVIANTFQVIVSQRVRQVALLRLVGGHRRQVSRVVLAEAA IAGSIGAVIGAVAGVGLGYLGAGLLDINGGGLAVNPIVLALCVLTGVGAT VVAAWAPARRATRVPPVRALQEVPDALPAQVRGGRRLVAGLILIGLAVGV LGLAAIGTSLPLALVGGVLLAAGLLTALPLGIALLLPPAARGLERFGVAA SLAGSNLRQNAR

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biochemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Engineering & Computer Science (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Public Health (AREA)
  • Biophysics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • General Chemical & Material Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Virology (AREA)
  • Biomedical Technology (AREA)
  • Microbiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Communicable Diseases (AREA)
  • Oncology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
PCT/GB2007/000138 2006-01-17 2007-01-17 Lantibiotic biosynthetic gene clusters from a. garbadinensis and a. liguriae WO2007083112A2 (en)

Priority Applications (16)

Application Number Priority Date Filing Date Title
PL07704921T PL1979375T3 (pl) 2006-01-17 2007-01-17 Klastry biosyntetycznych genów kodujących lantybiotyki pochodzące od a. garbadinensis oraz a. liguriae
ES07704921T ES2378090T3 (es) 2006-01-17 2007-01-17 Clústeres de genes biosintéticos lantibióticos a partir de A. garbadinensis y A. liguriae
JP2008550839A JP5219837B2 (ja) 2006-01-17 2007-01-17 Ａ．ガルバジネンシスおよびａ．リグリエに由来するランチビオティック生合成遺伝子クラスター
US13/705,010 USRE45003E1 (en) 2006-01-17 2007-01-17 Lantibiotic biosynthetic gene clusters from A. garbadinensis and A. liguriae
NZ569486A NZ569486A (en) 2006-01-17 2007-01-17 Lantibiotic biosynthetic gene clusters from A.garbadinensis and A.liguriae
BRPI0706525-6A BRPI0706525A2 (pt) 2006-01-17 2007-01-17 grupamentos genéticos biossintéticos de lantiobióticos de a. garbadinensis e a. liguriae
DK07704921.1T DK1979375T3 (da) 2006-01-17 2007-01-17 Lantibiotiske, biosyntetiske genklynger fra A. garbadinensis og A. liguriae
US12/161,221 US7989416B2 (en) 2006-01-17 2007-01-17 Lantibiotic biosynthetic gene clusters from A. garbadinensis and A. Liguriae
MX2008009047A MX2008009047A (es) 2006-01-17 2007-01-17 Grupos de genes biosinteticos lantibioticos de a. garbadinesis y a. liguriae.
CN2007800067480A CN101389641B (zh) 2006-01-17 2007-01-17 来自菌株产加尔巴丁游动放线菌和利古里亚游动放线菌中的羊毛硫细菌素的生物合成成簇基因
EP07704921A EP1979375B9 (en) 2006-01-17 2007-01-17 Lantibiotic biosynthetic gene clusters from a. garbadinensis and a. liguriae
CA002637315A CA2637315A1 (en) 2006-01-17 2007-01-17 Lantibiotic biosynthetic gene clusters from a. garbadinensis and a. liguriae
AU2007206769A AU2007206769B2 (en) 2006-01-17 2007-01-17 Lantibiotic biosynthetic gene clusters from A. garbadinensis and A. liguriae
AT07704921T ATE534659T1 (de) 2006-01-17 2007-01-17 Biosynthetische gencluster für lantibiotikum aus a. garbadinensis und a. liguriae
IL192446A IL192446A0 (en) 2006-01-17 2008-06-25 Lantibiotic biosynthetic gene clusters form a. garbadinensis and a. liguriae
US13/157,454 US8465947B2 (en) 2006-01-17 2011-06-10 Lantibiotic biosynthetic gene clusters from A. garbadinensis and A. liguriae

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GBGB0600928.6A GB0600928D0 (en) 2006-01-17 2006-01-17 Improvements relating to lantibiotics
GB0600928.6 2006-01-17

Related Child Applications (2)

Application Number Title Priority Date Filing Date
US12/161,221 A-371-Of-International US7989416B2 (en) 2006-01-17 2007-01-17 Lantibiotic biosynthetic gene clusters from A. garbadinensis and A. Liguriae
US13/157,454 Continuation US8465947B2 (en) 2006-01-17 2011-06-10 Lantibiotic biosynthetic gene clusters from A. garbadinensis and A. liguriae

Publications (2)

Publication Number Publication Date
WO2007083112A2 true WO2007083112A2 (en) 2007-07-26
WO2007083112A3 WO2007083112A3 (en) 2008-05-08

Family

ID=35998187

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB2007/000138 WO2007083112A2 (en) 2006-01-17 2007-01-17 Lantibiotic biosynthetic gene clusters from a. garbadinensis and a. liguriae

Country Status (20)

Country Link
US (3) US7989416B2 (US07794700-20100914-C00152.png)
EP (2) EP1979375B9 (US07794700-20100914-C00152.png)
JP (1) JP5219837B2 (US07794700-20100914-C00152.png)
KR (1) KR101573536B1 (US07794700-20100914-C00152.png)
CN (1) CN101389641B (US07794700-20100914-C00152.png)
AT (1) ATE534659T1 (US07794700-20100914-C00152.png)
AU (1) AU2007206769B2 (US07794700-20100914-C00152.png)
BR (1) BRPI0706525A2 (US07794700-20100914-C00152.png)
CA (1) CA2637315A1 (US07794700-20100914-C00152.png)
CY (1) CY1112559T1 (US07794700-20100914-C00152.png)
DK (1) DK1979375T3 (US07794700-20100914-C00152.png)
ES (1) ES2378090T3 (US07794700-20100914-C00152.png)
GB (1) GB0600928D0 (US07794700-20100914-C00152.png)
IL (1) IL192446A0 (US07794700-20100914-C00152.png)
MX (1) MX2008009047A (US07794700-20100914-C00152.png)
NZ (1) NZ569486A (US07794700-20100914-C00152.png)
PL (1) PL1979375T3 (US07794700-20100914-C00152.png)
PT (1) PT1979375E (US07794700-20100914-C00152.png)
SI (1) SI1979375T1 (US07794700-20100914-C00152.png)
WO (1) WO2007083112A2 (US07794700-20100914-C00152.png)

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009010763A1 (en) * 2007-07-18 2009-01-22 Novacta Biosystems Limited The use of type-b lantibiotic-based compounds having antimicrobial activity
WO2010082019A1 (en) 2009-01-14 2010-07-22 Novacta Biosystems Limited Actagardine derivatives, and pharmaceutical use thereof
WO2010082018A1 (en) 2009-01-14 2010-07-22 Novacta Biosystems Limited Deoxyactagardine derivatives
WO2010089544A1 (en) 2009-02-04 2010-08-12 Novacta Biosystems Limited Actagardine derivatives
US7989416B2 (en) 2006-01-17 2011-08-02 Novacta Biosystems Limited Lantibiotic biosynthetic gene clusters from A. garbadinensis and A. Liguriae
WO2011095769A1 (en) 2010-02-02 2011-08-11 Novacta Biosystems Limited Actagardine derivatives, and pharmaceutical use thereof
WO2011095768A1 (en) 2010-02-02 2011-08-11 Novacta Biosystems Limited Lantibiotic salts
WO2012007711A1 (en) 2010-07-14 2012-01-19 Novacta Biosystems Limited Formulation comprising a type b lantibiotic
WO2012020222A1 (en) 2010-08-11 2012-02-16 Novacta Biosystems Limited Compounds
WO2012020221A1 (en) 2010-08-11 2012-02-16 Novacta Biosystems Limited Compounds
WO2012020220A1 (en) 2010-08-11 2012-02-16 Novacta Biosystems Limited Compounds
WO2012020219A3 (en) * 2010-08-11 2012-04-05 Novacta Biosystems Limited Formulations for infusion of type b lantibiotics
US8329644B2 (en) 2007-07-18 2012-12-11 Novacta Biosystems Limited Lantibiotic-based compounds having antimicrobial activity
CN103483433A (zh) * 2013-09-10 2014-01-01 中国科学院微生物研究所 一种新型高效的羊毛硫细菌素cerecidin及其应用

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9975930B2 (en) 2012-11-30 2018-05-22 Naicons S.R.L. Lantibiotic derivatives and process for their preparation
CN106188253B (zh) * 2016-08-26 2020-08-18 上海交通大学 抗菌肽Lexapeptide及其制备方法和用途

Family Cites Families (37)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3710795A (en) 1970-09-29 1973-01-16 Alza Corp Drug-delivery device with stretched, rate-controlling membrane
GB8507528D0 (en) 1985-03-22 1985-05-01 Lepetit Spa Basis monocarboxyamide derivatives
US5304540A (en) * 1988-06-22 1994-04-19 Applied Microbiology, Inc. Pharmaceutical bacteriocin compositions and methods for using the same
IN167138B (US07794700-20100914-C00152.png) 1988-08-17 1990-09-01 Hoechst India
GB8926639D0 (en) 1989-11-24 1990-01-17 Agricultural & Food Res Delayed release formulations
US5376645A (en) 1990-01-23 1994-12-27 University Of Kansas Derivatives of cyclodextrins exhibiting enhanced aqueous solubility and the use thereof
KR0166088B1 (ko) 1990-01-23 1999-01-15 . 수용해도가 증가된 시클로덱스트린 유도체 및 이의 용도
IT1260505B (it) 1992-06-01 1996-04-09 Poli Ind Chimica Spa Sistemi farmaceutici orali a cessione ritardata per il rilascio specifico nel colon
IL107887A (en) * 1992-12-08 2003-07-06 Ambi Inc Stabilized lanthionine containing bacteriocin compositions
US5512269A (en) 1993-06-09 1996-04-30 Burroughs Wellcome, Co. Method of treating retained pulmonary secretions
PT700998E (pt) 1994-09-12 2004-03-31 Aventis Pharma Gmbh Mersacidina recombinante e metodo para a sua producao
JP4236699B2 (ja) 1995-06-23 2009-03-11 アンビィー インコーポレイテッド 抗生物質抵抗性グラム陽性細菌の制御および感染治療の方法
GB9711643D0 (en) 1997-06-05 1997-07-30 Janssen Pharmaceutica Nv Glass thermoplastic systems
US5985823A (en) 1997-06-09 1999-11-16 Ambi Inc. Method for the treatment of diarrheal disease and for eliminating particular bacterial populations from the colon
US5958873A (en) 1997-06-09 1999-09-28 University Of Cincinnati Oral formulation for treatment of bacteria-induced diseases of the colon
DE19745583A1 (de) * 1997-10-15 1999-04-22 Hoechst Marion Roussel De Gmbh Neues Lantibiotikum verwandt mit Actagardine, Verfahren zur Herstellung und Verwendung derselben
US6569830B1 (en) 1999-03-05 2003-05-27 Ambi, Inc. Compositions and methods for treatment of staphylococcal infection while suppressing formation of antibiotic-resistant strains
PT1294358E (pt) 2000-06-28 2004-12-31 Smithkline Beecham Plc Processo de moagem por via humida
GB0110432D0 (en) * 2001-04-27 2001-06-20 Plant Bioscience Ltd Lantibiotic production
WO2002103010A1 (en) 2001-06-14 2002-12-27 Plant Bioscience Limited Methods and materials for targeted gene disruption in actinomycete bacteria
US6861236B2 (en) 2002-05-24 2005-03-01 Applied Nanosystems B.V. Export and modification of (poly)peptides in the lantibiotic way
WO2004033706A2 (en) 2002-10-10 2004-04-22 Molichem Medicines, Inc. Nucleic acids encoding duramycin
US7351687B2 (en) 2003-07-18 2008-04-01 Vicuron Pharmaceuticals, Inc. Antibiotic 107891, its factors A1 and A2, pharmaceutically acceptable salts and compositions, and use thereof
WO2005014628A1 (en) 2003-07-18 2005-02-17 Vicuron Pharmaceuticals Inc. Antibiotic 107891, its factors a1 and a2, pharmaceutically acceptable salts and compositions, and use thereof.
WO2005079508A2 (en) * 2004-02-17 2005-09-01 Cancervax Corporation Method and composition for angiogenesis inhibition
GB0406870D0 (en) 2004-03-26 2004-04-28 Novacta Biosystems Ltd Improvements relating to the production of lantibiotics
CA2623624A1 (en) 2005-09-27 2007-04-05 Novacta Biosystems Limited Variants of the lantibiotic mersacidin and their use
GB0600928D0 (en) 2006-01-17 2006-02-22 Novacta Biosystems Ltd Improvements relating to lantibiotics
AU2007310534A1 (en) 2006-10-27 2008-05-02 Capsugel Belgium Nv Hydroxypropyl methyl cellulose hard capsules and process of manufacture
CA2690267A1 (en) 2007-06-12 2008-12-18 The University Of British Columbia Small cationic antimicrobial peptides
GB0714029D0 (en) 2007-07-18 2007-08-29 Novacta Biosystems Ltd Lantibiotic-based compounds having antimicrobial activity
GB0714030D0 (en) 2007-07-18 2007-08-29 Novacta Biosystems Ltd The use of type-B lantibiotic-based compounds having antimicrobial activity
WO2010058238A1 (en) 2008-11-24 2010-05-27 Sentinella Pharmaceuticals, Inc. ("Sentinella") Lantibiotic carboxyamide derivatives with enhanced antibacterial activity
GB0900599D0 (en) 2009-01-14 2009-02-18 Novacta Biosystems Ltd Treatment
JP5719312B2 (ja) 2009-01-14 2015-05-13 ノヴァクタ バイオシステムズ リミティッド デオキシアクタガルジン誘導体
CA2750883A1 (en) 2009-02-04 2010-08-12 Novacta Biosystems Limited Actagardine derivatives
EP2216937A1 (en) 2009-02-10 2010-08-11 Alcatel Lucent Alarm notification between customer premises equipment and a remote management server

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
None

Cited By (39)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
USRE45003E1 (en) 2006-01-17 2014-07-08 Novacta Biosystems Limited Lantibiotic biosynthetic gene clusters from A. garbadinensis and A. liguriae
US7989416B2 (en) 2006-01-17 2011-08-02 Novacta Biosystems Limited Lantibiotic biosynthetic gene clusters from A. garbadinensis and A. Liguriae
US8465947B2 (en) 2006-01-17 2013-06-18 Novacta Biosystems Limited Lantibiotic biosynthetic gene clusters from A. garbadinensis and A. liguriae
US8329644B2 (en) 2007-07-18 2012-12-11 Novacta Biosystems Limited Lantibiotic-based compounds having antimicrobial activity
EA021628B1 (ru) * 2007-07-18 2015-07-30 Новакта Биосистемс Лимитед СОЕДИНЕНИЯ АКТАГАРДИНА И ИХ ПРИМЕНЕНИЕ ДЛЯ ЛЕЧЕНИЯ ИЛИ ПРОФИЛАКТИКИ ИНФЕКЦИИ Clostridium difficile
WO2009010763A1 (en) * 2007-07-18 2009-01-22 Novacta Biosystems Limited The use of type-b lantibiotic-based compounds having antimicrobial activity
US8575094B2 (en) 2007-07-18 2013-11-05 Novacta Biosystems Limited Use of type-B lantibiotic-based compounds having antimicrobial activity
US8741945B2 (en) * 2009-01-14 2014-06-03 Novacta Biosystems Limited Compounds
US20120277145A1 (en) * 2009-01-14 2012-11-01 Sjoerd Nicolaas Wadman Compounds
EA021683B1 (ru) * 2009-01-14 2015-08-31 Новакта Биосистемс Лимитед Производные деоксиактагардина в
WO2010082019A1 (en) 2009-01-14 2010-07-22 Novacta Biosystems Limited Actagardine derivatives, and pharmaceutical use thereof
TWI453029B (zh) * 2009-01-14 2014-09-21 Novacta Biosystems Ltd 去氧艾克特卡丁b(deoxyactagardine b), 包含其之醫藥組合物及其製造方法
WO2010082018A1 (en) 2009-01-14 2010-07-22 Novacta Biosystems Limited Deoxyactagardine derivatives
JP2012515195A (ja) * 2009-01-14 2012-07-05 ノヴァクタ バイオシステムズ リミティッド デオキシアクタガルジン誘導体
AU2010205472B2 (en) * 2009-01-14 2013-03-14 Novacta Biosystems Limited Deoxyactagardine derivatives
US8283371B2 (en) * 2009-01-14 2012-10-09 Novacta Biosystems Limited Compounds
AU2010212183B2 (en) * 2009-02-04 2014-07-10 Novacta Biosystems Limited Actagardine derivatives
EA020733B1 (ru) * 2009-02-04 2015-01-30 Новакта Биосистемс Лимитед Производные актагардина
JP2012516883A (ja) * 2009-02-04 2012-07-26 ノヴァクタ バイオシステムズ リミティッド アクタガルジン誘導体
CN102388060A (zh) * 2009-02-04 2012-03-21 诺瓦克塔生物系统有限公司 阿肽加定衍生物
CN102388060B (zh) * 2009-02-04 2014-09-10 诺瓦克塔生物系统有限公司 阿肽加定衍生物
WO2010089544A1 (en) 2009-02-04 2010-08-12 Novacta Biosystems Limited Actagardine derivatives
US8729031B2 (en) 2009-02-04 2014-05-20 Novacta Biosystems Limited Compounds
JP2013518867A (ja) * 2010-02-02 2013-05-23 ノヴァクタ バイオシステムズ リミティッド ランチビオティックの塩
WO2011095769A1 (en) 2010-02-02 2011-08-11 Novacta Biosystems Limited Actagardine derivatives, and pharmaceutical use thereof
US9006392B2 (en) 2010-02-02 2015-04-14 Novacta Biosystems Limited Actagardine derivatives, and pharmaceutical use thereof
WO2011095768A1 (en) 2010-02-02 2011-08-11 Novacta Biosystems Limited Lantibiotic salts
WO2012007711A1 (en) 2010-07-14 2012-01-19 Novacta Biosystems Limited Formulation comprising a type b lantibiotic
CN103108628A (zh) * 2010-08-11 2013-05-15 诺瓦克塔生物系统有限公司 用于输注b型羊毛硫抗生素的制剂
WO2012020222A1 (en) 2010-08-11 2012-02-16 Novacta Biosystems Limited Compounds
WO2012020219A3 (en) * 2010-08-11 2012-04-05 Novacta Biosystems Limited Formulations for infusion of type b lantibiotics
JP2013533304A (ja) * 2010-08-11 2013-08-22 ノヴァクタ バイオシステムズ リミティッド Ｂ型ランチビオティックの点滴用製剤
US20130137630A1 (en) * 2010-08-11 2013-05-30 Novacta Biosystems Limited Formulations for infusion of type b lantibiotics
WO2012020220A1 (en) 2010-08-11 2012-02-16 Novacta Biosystems Limited Compounds
WO2012020221A1 (en) 2010-08-11 2012-02-16 Novacta Biosystems Limited Compounds
US9192569B2 (en) 2010-08-11 2015-11-24 Novacta Biosystems Limited Formulations for infusion of type B lantibiotics
EA024344B1 (ru) * 2010-08-11 2016-09-30 Новакта Биосистемс Лимитед Составы для инфузии лантибиотиков типа b
CN103483433A (zh) * 2013-09-10 2014-01-01 中国科学院微生物研究所 一种新型高效的羊毛硫细菌素cerecidin及其应用
CN103483433B (zh) * 2013-09-10 2015-05-27 中国科学院微生物研究所 一种新型高效的羊毛硫细菌素cerecidin及其应用

Also Published As

Publication number Publication date
JP5219837B2 (ja) 2013-06-26
KR20080086517A (ko) 2008-09-25
PT1979375E (pt) 2012-03-06
CN101389641B (zh) 2013-06-05
US20100048459A1 (en) 2010-02-25
EP2189472A1 (en) 2010-05-26
NZ569486A (en) 2011-07-29
DK1979375T3 (da) 2012-03-19
WO2007083112A3 (en) 2008-05-08
ATE534659T1 (de) 2011-12-15
GB0600928D0 (en) 2006-02-22
US7989416B2 (en) 2011-08-02
EP1979375B9 (en) 2012-05-09
MX2008009047A (es) 2008-12-18
AU2007206769A1 (en) 2007-07-26
BRPI0706525A2 (pt) 2011-03-29
AU2007206769B2 (en) 2013-06-20
CA2637315A1 (en) 2007-07-26
CY1112559T1 (el) 2016-02-10
EP1979375A2 (en) 2008-10-15
IL192446A0 (en) 2008-12-29
US8465947B2 (en) 2013-06-18
US20110306091A1 (en) 2011-12-15
JP2009523777A (ja) 2009-06-25
SI1979375T1 (sl) 2012-03-30
PL1979375T3 (pl) 2012-04-30
ES2378090T3 (es) 2012-04-04
CN101389641A (zh) 2009-03-18
KR101573536B1 (ko) 2015-12-11
EP1979375B1 (en) 2011-11-23
USRE45003E1 (en) 2014-07-08

Similar Documents

Publication Publication Date Title
US7989416B2 (en) Lantibiotic biosynthetic gene clusters from A. garbadinensis and A. Liguriae
US20140038297A1 (en) Genes and Proteins for the Biosynthesis of the Lantibiotic 107891
WO1999005283A2 (fr) Genes de biosynthese et de transfert des 6-desoxyhexoses chez saccharopolyspora erythraea et chez streptomyces antibioticus et leur utilisation
CA2257111A1 (en) Isolation of the biosynthesis genes for pseudo-oligosaccharides from streptomyces glaucescens gla.o and their use
US20080145892A1 (en) Genes and Proteins For the Biosynthesis of the Glycopeptide Antibiotic A40926
WO2006111743A2 (en) Identifying lantibiotic gene clusters and novel lantibiotic genes
RU2441915C2 (ru) Гены и белки для биосинтеза лантибиотика 107891
Spohn Exploiting gene regulation as an approach to identify, analyze and utilize the biosynthetic pathways of the glycopeptide ristomycin A and the zincophore [S, S]-EDDS in Amycolatopsis japonicum
Bell Analysis and manipulation of “actagardine” gene clusters from Actinoplanes
KR20050084843A (ko) 스피라마이신의 생합성에 관여하는 폴리펩티드, 이들폴리펩티드를 암호화하는 뉴클레오티드 서열 및 이의 용도
FR2766496A1 (fr) Genes de biosynthese et de transfert des 6-desoxyhexoses chez saccharopolyspora erythraea et leur utilisation

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application
WWE Wipo information: entry into national phase

Ref document number: 2007206769

Country of ref document: AU

WWE Wipo information: entry into national phase

Ref document number: 569486

Country of ref document: NZ

WWE Wipo information: entry into national phase

Ref document number: MX/a/2008/009047

Country of ref document: MX

WWE Wipo information: entry into national phase

Ref document number: 2637315

Country of ref document: CA

Ref document number: 2008550839

Country of ref document: JP

NENP Non-entry into the national phase

Ref country code: DE

WWE Wipo information: entry into national phase

Ref document number: 1020087018136

Country of ref document: KR

ENP Entry into the national phase

Ref document number: 2007206769

Country of ref document: AU

Date of ref document: 20070117

Kind code of ref document: A

WWP Wipo information: published in national office

Ref document number: 2007206769

Country of ref document: AU

WWE Wipo information: entry into national phase

Ref document number: 2007704921

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 3225/KOLNP/2008

Country of ref document: IN

WWE Wipo information: entry into national phase

Ref document number: 200780006748.0

Country of ref document: CN

WWE Wipo information: entry into national phase

Ref document number: 12161221

Country of ref document: US

Ref document number: 13705010

Country of ref document: US

ENP Entry into the national phase

Ref document number: PI0706525

Country of ref document: BR

Kind code of ref document: A2

Effective date: 20080714