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 (AICE). 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:
[0001]    The prokaryotic organism  Actinoplanes  sp. SE50/110 produces the alpha-glucosidase inhibitor acarbose, which is used worldwide in the treatment of diabetes mellitus type-2. Based on the fact, that the incidence of diabetes type-2 is rapidly rising worldwide, an increasing demand for acarbose is expected in the future. In order to meet these expectations, genetic manipulations of the strain and its derivatives have to be carried out, aiming at increasing acarbose yields. However, currently no tools for genetic manipulation exist for this strain, hampering the process of strain improvement. 
         [0002]    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 (AICE). 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 OF THE INVENTION 
       [0003]      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). 
         [0004]    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). 
         [0005]    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 ( FIG. 1 ). 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). 
     
    
     
         [0006]      FIG. 1  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). 
           [0007]      FIG. 2  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. 
       
    
    
       [0008]    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). 
         [0009]    Besides the proline tRNA genomic integration site, pACPL was shown to subsist in at least twelve copies ( FIG. 2 ) as an extrachromosomal element in an average  Actinoplanes  sp. SE50/110 cell (Schwientek et al., 2012). pACPL hosts 22 protein coding sequences. 
         [0010]    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.       
 
         [0014]    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.       
 
         [0018]    The most preferred product in this method is acarbose. 
       DETAILED DESCRIPTION OF THE 22 PROTEIN CODING SEQUENCES OF pACPL 
       [0019]    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). 
         [0020]    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). 
         [0021]    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). 
         [0022]    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. 
         [0023]    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). 
         [0024]    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. 
         [0025]    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. 
         [0026]    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. 
         [0027]    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. 
         [0028]    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). 
         [0029]    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. 
         [0030]    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. 
         [0031]    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). 
         [0032]    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. 
         [0033]    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. 
         [0034]    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. 
         [0035]    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. 
         [0036]    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). 
         [0037]    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. 
         [0038]    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). 
         [0039]    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. 
         [0040]    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. 
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