Patent Application: US-201214363810-A

Abstract:
the present invention is directed to an innate dna sequence within the complete genome sequence of actinoplanes sp . se50 / 110 which resembles the structure of an actinomycete integrative and conjugative element . related aices were used for establishing genetic manipulation tools for other bacteria in the past . in this document , we describe the unique features of the specific aice found in actinoplanes sp . se50 / 110 which are clearly distinct from any other known aice as a whole , but share minor parts with varying sequence similarity with other characterized aices from other species .

Description:
actinoplanes sp . se50 / 110 is a gram - positive , aerobic bacterium with a high g + c content genome of about 9 . 25 mb in size ( schwientek et al ., 2012 ). the medically important organism is the natural producer of a variety of chemically related substances , which were found to inhibit human alpha - glucosidases ( caspary and graf , 1979 ), making them especially suitable for pharmaceutical applications ( frommer et al ., 1975 , 1977 a , 1977 b , 1979 ). in particular , the pseudotetrasaccharide acarbose , which is synthesized through enzymes encoded in the well characterized acarbose gene cluster ( wehmeier and piepersberg , 2004 ), is used worldwide in the treatment of type - 2 diabetes mellitus ( non - insulin - dependent ). diabetes mellitus type - 2 is a chronic disease with more than 250 million people affected worldwide . inappropriately managed or untreated , it can lead to severe cases of renal failure , blindness , slowly healing wounds and arterial diseases , including coronary artery atherosclerosis ( idf , 2009 ). as the incidence of diabetes type 2 is rapidly rising worldwide , an ever increasing demand for diabetes drugs like acarbose needs to be anticipated . the pseudotetrasaccharide acarbose is currently produced by industrial fermentation of yield - optimized strains , which are based on the wild - type bacterium actinoplanes sp . se50 / 110 ( atcc 31044 ; cbs 674 . 73 ). while classical strain optimization through conventional mutagenesis was a very successful way of increasing the production of acarbose in the past , this strategy seems to have reached its limits by now . in order to further increase production efficacy , targeted genetic engineering methods have to be applied , which requires a functional transformation system for actinoplanes sp . se50 / 110 . previous experiments revealed that actinoplanes sp . se50 / 110 and actinoplanes friuliensis ( and presumably most other actinoplanes spp .) do not allow for standard transformation methods like electroporation or peg - mediated transformation , despite serious efforts have been made ( heinzelmann et al ., 2003 ). in this context , an actinomycete integrative and conjugative element ( aice ) has been identified on the actinoplanes sp . se50 / 110 genome ( genbank : cp003170 ), which can be used for this purpose as has been shown previously for related species ( hosted et al ., 2005 ). aices are a class of mobile genetic elements possessing a highly conserved structural organization with functional modules for excision / integration , replication , conjugative transfer and regulation ( te poele , bolhuis , et al ., 2008 ). being able to replicate autonomously , they are also said to mediate the acquisition of additional modules encoding functions , such as resistance and metabolic traits , which confer a selective advantage to the host under certain environmental conditions ( burrus and waldor , 2004 ). a new aice , designated pacpl , was identified in the complete genome sequence of actinoplanes sp . se50 / 110 ( fig1 ). its size of 13 . 6 kb and the structural gene organization are in good accordance with other known aices of closely related species like micromonospora rosario , salinispora tropica or streptomyces coelicolor ( te poele , bolhuis , et al ., 2008 ). fig1 structural organization of the newly identified actinomycete integrative and conjugative element ( aice ) pacpl from actinoplanes sp . se50 / 110 . typical genes that are also found on other aices are colored : excision / integration ( orange ), replication ( yellow ), main transfer ( dark blue ), conjugation ( blue ), nudix hydrolase ( dark green ), regulation ( green ), other annotated function ( red ), unknown function ( gray ). fig2 scatter plot of 571 actinoplanes sp . se50 / 110 contigs resulting from automatic combined assembly of paired end and whole genome shotgun pyrosequencing runs . the average number of reads per base is 21 . 12 and is depicted in the plot by the central diagonal line marked with ‘ average ’. additional lines indicate the factor of over - and underrepresentation of reads per base up to a factor of 10 and 1 / 10 fold , respectively . the axes represent logarithmic scales . large and highly overrepresented contigs are highlighted by special symbols . each contig is represented by one of the following symbols : diamond , regular contig ; square , contig related to the actinomycete integrative and conjugative element ( aice ); triangle , contig related to ribosomal operon ( rrn ); circle , related to transposons . most known aices subsist in their host genome by integration in the 3 ′ end of a trna gene by site - specific recombination between two short identical sequences ( att identity segments ) within the attachment sites located on the genome ( attb ) and the aice ( attp ), respectively ( te poele , bolhuis , et al ., 2008 ). in pacpl , the att identity segments are 43 nt in size and attb overlaps the 3 ′ end of a proline trna gene . moreover , the identity segment in attp is flanked by two 21 nt repeats containing two mismatches : gtcacccagttagt ( t / c ) ac ( c / t ) cag . these exhibit high similarities to the arm - type sites identified in the aice psam2 from strepomyces ambofaciens . for psam2 it was shown that the integrase binds to these repeats and that they are essential for efficient recombination ( raynal et al ., 2002 ). besides the proline trna genomic integration site , pacpl was shown to subsist in at least twelve copies ( fig2 ) as an extrachromosomal element in an average actinoplanes sp . se50 / 110 cell ( schwientek et al ., 2012 ). pacpl hosts 22 protein coding sequences . the actinomycete integrative and conjugative element of the present invention is selected from the group consisting of : a ) a polynucleotide having the sequence of seq id 1 , b ) a polynucleotide which hybridizes under stringent conditions to a polynucleotide as specified in ( a ) and c ) a polynucleotide having at least 90 % identity with the sequence of seq id 1 . preferred are aices having at least 95 % identity with the sequence of seq id 1 . more preferred are aices having at least 98 % identity with the sequence of seq id 1 . the present invention is further related to a host cell that has been transformed with the actinomycete integrative and conjugative element described above . the most preferred host cell is an actinoplanes sp . the host cell is useful in a method for preparation of biological products comprising the steps of a ) culturing the above host cell in a useful medium , b ) harvesting the product from the culture and c ) isolating and purifying the product . the gene int ( genomic locus tag : acpl — 6310 ) encodes the integrase of the aice with a length of 388 amino acids . its sequence shows 74 % similarity to an integrase ( genbank : efl40120 . 1 ) of streptomyces griseoflavus tu4000 within the first 383 amino acids . the integrase domain of the protein is located from amino acid 182 - 365 and shows high similarity ( e - value 2 . 90e - 21 ) to the int / topo ib signature motif ( conserved domain : cd01182 ). the integrase is responsible for integration into a trna gene by site - specific recombination which occurs between the two similar attachment sites attb on the chromosome and attp on the aice ( te poele , bolhuis , et al ., 2008 ). the gene xis ( genomic locus tag : acpl — 6309 ) encodes the excisionase of the aice with a length of 68 amino acids ). it shows highest similarity to the hypothetical protein sros — 7036 ( genbank : acz89735 . 1 ) from streptosporangium roseum dsm 43021 . the protein contains a moderately conserved ( e - value : 1 . 31e - 07 ) helix - turn - helix motif ( pfam12728 ) between amino acids 9 - 55 . xis is needed in combination with int to mediate the excision of the aice from the chromosome in preparation for amplification and transfer to other hosts ( te poele , bolhuis , et al ., 2008 ). the gene repsa ( genomic locus tag : acpl — 6308 ) encodes the replication initiation protein of the aice with a length of 598 amino acids . it has highest similarity to a putative plasmid replication initiation protein ( genbank : adl48867 . 1 ) from micromonospora aurantiaca atcc 27029 . the protein resembles the well characterized repsa protein from streptomyces ambofaciens which has been found to apply a rolling cycle replication mechanism ( hagège et al ., 1993 ). the gene aice1 ( genomic locus tag : acpl — 6307 ) encodes a protein with unknown function with a length of 97 amino acids . it shows 69 % similarity in the first 80 amino acids to the hypothetical protein micau — 5360 ( genbank : adl48866 . 1 ) from micromonospora aurantiaca atcc 27029 . the gene spda ( genomic locus tag : acpl — 6306 ) encodes a putative spread protein of the aice with a length of 107 amino acids . spda shows 54 % similarity to a spread protein ( genbank : abd10289 . 1 ) from frankia sp . cci3 . spread proteins are involved in pock formation , which reflects a temporary growth delay of recipient cells that are in the process of acquiring an aice from a donor cell . thus , spread proteins assist in the intramycelial spread of ( kataoka et al ., 1994 ; grohmann et al ., 2003 ; te poele , bolhuis , et al ., 2008 ). the gene spdb ( genomic locus tag : acpl — 6305 ) encodes a putative spread protein of the aice with a length of 169 amino acids . spdb shows 84 % similarity between the amino acids 40 - 131 to a spread protein ( genbank : aax38998 . 1 ) from micromonospora rosaria . spread proteins are involved in pock formation , which reflects a temporary growth delay of recipient cells that are in the process of acquiring an aice from a donor cell . thus , spread proteins assist in the intramycelial spread of ( kataoka et al ., 1994 ; grohmann et al ., 2003 ; te poele , bolhuis , et al ., 2008 ). a signal peptide has been found for spdb , its cleavage site is predicted at position 18 . furthermore , three transmembrane helices were found at positions i53 - 70o75 - 97i109 - 131o . the gene aice2 ( genomic locus tag : acpl — 6304 ) encodes a protein with unknown function with a length of 96 amino acids . it shows 57 % similarity between the amino acids 12 - 89 to the hypothetical protein micau — 5358 ( genbank : adl48864 . 1 ) from micromonospora aurantiaca atcc 27029 . the gene aice3 ( genomic locus tag : acpl — 6303 ) encodes a protein with unknown function with a length of 61 amino acids . it shows no significant similarity to any of the proteins in public databases . the gene aice4 ( genomic locus tag : acpl — 6302 ) encodes a protein with unknown function with a length of 138 amino acids . it shows 69 % similarity in the last 113 amino acids to the hypothetical protein micau — 5357 ( genbank : adl48863 . 1 ) from micromonospora aurantiaca atcc 27029 . the gene aice5 ( genomic locus tag : acpl — 6301 ) encodes a protein with unknown function with a length of 108 amino acids . it shows 79 % similarity to the complete amino acid sequence of the hypothetical protein micau — 5356 ( genbank : adl48862 . 1 ) from micromonospora aurantiaca atcc 27029 . this protein has a low pfam hit ( e - value 0 . 0022 ) to sigma factors with extracytoplasmic function ( ecf ). these sigma factors can bind to rna polymerase in order to stimulate the transcription of specific genes . they are believed to be activated upon receiving a stimulus from the environment and are often cotranscribed with one or more negative regulators ( heimann , 2002 ). the gene aice6 ( genomic locus tag : acpl — 6300 ) encodes a protein with unknown function with a length of 149 amino acids . it shows 50 % similarity to the complete amino acid sequence of the hypothetical protein vab18032 — 01645 ( genbank : aeb47413 . 1 ) from verrucosispora maris ab - 18 - 032 . the gene aice7 ( genomic locus tag : acpl — 6299 ) encodes a protein with unknown function with a length of 66 amino acids . it shows no similarity to any of the proteins in public databases . aice7 contains a single transmembrane helix ranging from amino acid 9 - 31 . the gene tra ( genomic locus tag : acpl — 6298 ) encodes the main transfer protein of the aice with a length of 293 amino acids . it exhibits 74 % similarity throughout the major part to a cell division protein ( genbank : adl48859 . 1 ) from micromonospora aurantiaca atcc 27029 . tra contains a domain with significant similarity ( e - value 3 . 1e - 14 ) to the ftsk / spollie domain between amino acids 29 - 187 , which is found in all aices and streptomyces transferase genes ( te poele , bolhuis , et al ., 2008 ). several experiments have provided evidence , that homologues of tra are responsible for the translocation of double - stranded dna to the recipient strains . translocation occurs at the hyphal tips of the mating mycelium ( possoz et al ., 2001 ; reuther et al ., 2006 ). the gene aice8 ( genomic locus tag : acpl — 6297 ) encodes a protein with unknown function with a length of 124 amino acids . it shows 44 % similarity between the amino acids 44 - 116 to the sequence of the fade6 protein ( genbank : egt86701 . 1 ) from mycobacterium colombiense cect 3035 . while the complete fade6 protein has 733 amino acids that resemble an acyl - coa dehydrogenase , aice8 is unlikely to have a similar function as it does not contain the catalytic domains of fade6 and is only 124 amino acids in length . the gene aice9 ( genomic locus tag : acpl — 6296 ) encodes a protein with unknown function with a length of 320 amino acids . it shows 68 % similarity throughout the major part of the sequence to the hypothetical protein micau — 5352 ( genbank : adl48858 . 1 ) from micromonospora aurantiaca atcc 27029 . this protein contains four transmembrane helices at positions 132 - 51o57 - 79i88 - 110o115 - 134i . the gene aice10 ( genomic locus tag : acpl — 6295 ) encodes a protein with unknown function with a length of 69 amino acids . it shows no significant similarity to any of the proteins in public databases . the gene pra ( genomic locus tag : acpl — 6294 ) is likely to encode the activator of the repsa , xis and int genes . it has a length of 105 amino acids and shows 90 % similarity throughout the complete sequence to the hypothetical protein micau — 5352 ( genbank : adl48857 . 1 ) from micromonospora aurantiaca atcc 27029 . pra , which regulates the transfer and replication of the aice , is believed to be repressed by the transcriptional regulator korsa in the aice psam2 from streptomyces ambofaciens ( sezonov et al ., 2000 ). by repressing pra , the aice remains in its integrated from on the chromosome . the gene reg ( genomic locus tag : acpl — 6293 ) encodes a regulatory protein of the aice with a length of 444 amino acids . it shows 50 % similarity throughout the complete sequence to a putative regulator ( genbank : ccb75999 . 1 ) from streptomyces cattleya nrrl 8057 . reg contains a helix - turn - helix domain , ranging from amino acids 4 - 72 . although the sequence similarity between reg and korsa from psam2 is very low , the localization of reg between the pra and nud genes may be an indication for reg resembling a homologue to korsa , which is frequently found in this genetic organization ( te poele , bolhuis , et al ., 2008 ). the gene nud ( genomic locus tag : acpl — 6292 ) encodes a protein which contains a nudix - hydrolase domain between amino acids 29 - 144 . it has a size of 172 amino acids and shows 72 % similarity throughout the sequence to a hypothetical protein ( genbank : efl09132 . 1 ) of streptomyces sp . aa4 and various nudix hydrolases from closely related species . nud exhibits 42 % similarity between amino acids 21 - 108 to the pif protein of psam2 . pif also contains a nudix - hydrolase domain , and was shown to be involved in intercellular signaling , which is believed to inhibit replication and transfer of the aice in order to prevent redundant transfer between psam2 harboring cells ( possoz et al ., 2003 ; te poele , bolhuis , et al ., 2008 ). it is therefore likely , that pra , reg and nud in pacpl resemble a similar regulatory mechanism like pra , korsa and pif do for psam2 . the gene mdp ( genomic locus tag : acpl — 6291 ) encodes a metal - dependent phosphohydrolase with a length of 80 amino acids . it exhibits 66 % similarity throughout its sequence to a metal - dependent phosphohydrolase ( genbank : abd10513 . 1 ) from frankia sp . cci3 . mdp encoding genes are frequently found in a cluster with pra , reg and nud homologues on other aices ( te poele , bolhuis , et al ., 2008 ). metal - dependent phosphohydrolases may be involved in signal transduction or nucleic acid metabolism ( te poele , samborskyy , et al ., 2008 ). the gene aice11 ( genomic locus tag : acpl — 6290 ) encodes a protein with unknown function with a length of 256 amino acids . it shows no significant similarity to any of the proteins in public databases . the gene aice12 ( genomic locus tag : acpl — 6289 ) encodes a protein with unknown function with a length of 93 amino acids . it shows no significant similarity to any of the proteins in public databases . burrus , v ., waldor , m . k ., 2004 . shaping bacterial genomes with integrative and conjugative elements . res . microbiol 155 , 376 - 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