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Mark J. Buttner - One of the best experts on this subject based on the ideXlab platform.
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phage p1 derived Artificial Chromosomes facilitate heterologous expression of the fk506 gene cluster
PLOS ONE, 2013Co-Authors: Adam C. Jones, Andreas Kulik, Bertolt Gust, Lutz Heide, Mark J. ButtnerAbstract:We describe a procedure for the conjugative transfer of phage P1-Derived Artificial Chromosome (PAC) library clones containing large natural product gene clusters (≥70 kilobases) to Streptomyces coelicolor strains that have been engineered for improved heterologous production of natural products. This approach is demonstrated using the gene cluster for FK506 (tacrolimus), a clinically important immunosuppressant of high commercial value. The entire 83.5 kb FK506 gene cluster from Streptomyces tsukubaensis NRRL 18488 present in one 130 kb PAC clone was introduced into four different S. coelicolor derivatives and all produced FK506 and smaller amounts of the related compound FK520. FK506 yields were increased by approximately five-fold (from 1.2 mg L-1 to 5.5 mg L-1) in S. coelicolor M1146 containing the FK506 PAC upon over-expression of the FK506 LuxR regulatory gene fkbN. The PAC-based gene cluster conjugation methodology described here provides a tractable means to evaluate and manipulate FK506 biosynthesis and is readily applicable to other large gene clusters encoding natural products of interest to medicine, agriculture and biotechnology.
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Phage P1-Derived Artificial Chromosome (PAC)-based approach for heterologous expression of FK506.
2013Co-Authors: Adam C. Jones, Bertolt Gust, Lutz Heide, Mark J. ButtnerAbstract:A) Structures of FK506 and FK520. B) Vector map of PAC pESAC13. C) Region of Streptomyces tsukubaensis NRRL 18488 genome included in PAC20N and used for heterologous expression. Gene colors refer to the following functions [30]: Yellow, allylmalonyl-CoA biosynthesis; light blue, methoxymalonyl-ACP biosynthesis; dark blue, polyketide synthase; green, chorismate hydrolase (starter unit biosynthesis); brown, peptide synthetase; orange, post-PKS modification; purple, FK506 regulation; red, thioesterase; black, other genes.
Manfred Schwab - One of the best experts on this subject based on the ideXlab platform.
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smallest region of overlapping deletion in 1p36 in human neuroblastoma a 1 mbp cosmid and pac contig
Genes Chromosomes and Cancer, 2001Co-Authors: Anja Bauer, Larissa Savelyeva, Andreas Claas, Christian Praml, Frank Berthold, Manfred SchwabAbstract:In human neuroblastomas, the distal portion of 1p is frequently deleted, as if one or more tumor suppressor genes from this region were involved in neuroblastoma tumorigenesis. Earlier studies had identified a smallest region of overlapping deletion (SRO) spanning approximately 23 cM between the most distally retained D1S80 and by the proximally retained D1S244. In pursuit of generating a refined delineation of the minimally deleted region, we have analyzed 49 neuroblastomas of different stages for loss of heterozygosity (LOH) from 1pter to 1p35 by employing 26 simple sequence length polymorphisms. Fifteen of the 49 tumors (31%) had LOH; homozygous deletion was not detected. Seven tumors had LOH at all informative loci analyzed, and eight tumors showed a terminal or an interstitial allelic loss of 1p. One small terminal and one interstitial deletion defined a new 1.7 cM SRO, approximately 1 Mbp in physical length, deleted in all tumors between the retained D1S2731 (distal) and D1S2666 (proximal). To determine the genomic complexity of the deleted region shared among tumors, we assembled a physical map of the I Mbp SRO consisting predominantly of bacteriophage P1-Derived Artificial Chromosome (PAC) clones. A total of 55 sequence-tagged site (STS) markers (23 published STSs and short tandem repeats and 32 newly identified STSs from the insert ends of PACs and cosmids) were assembled in a contig, resulting in a sequence-ready physical map with approximately one STS per 20 Kbp. Twelve genes (41BB, CD30, DFFA, DJ1, DR3, FRAP, HKR3, MASP2, MTHFR, RIZ, TNR2, TP73) previously mapped to 1p36 are localized outside this SRO. On the basis of this study, they would be excluded as candidate genes for neuroblastoma tumorigenesis. Ten expressed sequence tags were integrated in the contig, of which five are located outside the SRO. The other five from within the SRO may provide an entrance point for the cloning of candidate genes for neuroblastoma.
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smallest region of overlapping deletion in 1p36 in human neuroblastoma a 1 mbp cosmid and pac contig
Genes Chromosomes and Cancer, 2001Co-Authors: Anja Bauer, Larissa Savelyeva, Andreas Claas, Christian Praml, Frank Berthold, Manfred SchwabAbstract:In human neuroblastomas, the distal portion of 1p is frequently deleted, as if one or more tumor suppressor genes from this region were involved in neuroblastoma tumorigenesis. Earlier studies had identified a smallest region of overlapping deletion (SRO) spanning approximately 23 cM between the most distally retained D1S80 and by the proximally retained D1S244. In pursuit of generating a refined delineation of the minimally deleted region, we have analyzed 49 neuroblastomas of different stages for loss of heterozygosity (LOH) from 1pter to 1p35 by employing 26 simple sequence length polymorphisms. Fifteen of the 49 tumors (31%) had LOH; homozygous deletion was not detected. Seven tumors had LOH at all informative loci analyzed, and eight tumors showed a terminal or an interstitial allelic loss of 1p. One small terminal and one interstitial deletion defined a new 1.7 cM SRO, approximately 1 Mbp in physical length, deleted in all tumors between the retained D1S2731 (distal) and D1S2666 (proximal). To determine the genomic complexity of the deleted region shared among tumors, we assembled a physical map of the 1 Mbp SRO consisting predominantly of bacteriophage P1-Derived Artificial Chromosome (PAC) clones. A total of 55 sequence-tagged site (STS) markers (23 published STSs and short tandem repeats and 32 newly identified STSs from the insert ends of PACs and cosmids) were assembled in a contig, resulting in a sequence-ready physical map with approximately one STS per 20 Kbp. Twelve genes (41BB, CD30, DFFA, DJ1, DR3, FRAP, HKR3, MASP2, MTHFR, RIZ, TNR2, TP73) previously mapped to 1p36 are localized outside this SRO. On the basis of this study, they would be excluded as candidate genes for neuroblastoma tumorigenesis. Ten expressed sequence tags were integrated in the contig, of which five are located outside the SRO. The other five from within the SRO may provide an entrance point for the cloning of candidate genes for neuroblastoma. © 2001 Wiley-Liss, Inc.
Joseph J. Catanese - One of the best experts on this subject based on the ideXlab platform.
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a bacterial Artificial Chromosome library for sequencing the complete human genome
Genome Research, 2001Co-Authors: Kazutoyo Osoegawa, Eirik Frengen, Joseph J. Catanese, Pieter J. De Jong, Aaron G Mammoser, Changjiang ZengAbstract:The main goal of the publicly funded human genome project is to completely determine the human genomic DNA sequence. Five large centers in the United States and the United Kingdom (the G5 group) along with three smaller centers in France, Germany, and Japan (the G8 group) are the major contributors to the sequencing effort. The initial draft version of the human DNA sequence was completed on June 26, 2000, and a high-quality version will become accessible by 2003. The human genome project presents unique ethical and political requirements with respect to the source DNA for library construction, because never before has an individual's genetic blueprint been deciphered completely. One or more volunteers were required to donate their DNA for the sequencing effort. Donor recruitment must comply with regulations (Botkin and Gut 1996; Marshall 1996) to protect the individual's interests and requires informed consent. In addition, it is preferable to obtain the first human genome sequence with the focus on the composition of genes across the prototypical human genome rather than exploring the diversity of genes across the human population. With only a few donors contributing to the prototype of the human genome, it is likely that the prototype will not be equally derived from all ethnic or social groups. To avoid a willful bias with respect to representatives from one group or another, a double-blind donor selection protocol was desirable and was formulated in compliance with the stated policies of the funding agencies (see http://www.nhgri.nih.gov:80/Grant_info/Funding/Statements/RFA/human_subjects.html). Large-insert genomic DNA libraries in bacteria, such as bacterial Artificial Chromosome (BAC; Shizuya et al. 1992) and P1-Derived Artificial Chromosome (PAC; Ioannou et al. 1994) libraries, provide a way to divide the complexity of the human genome into a composite of large DNA segments of reduced complexity. Ideally, BAC libraries should completely represent the genome without cloning artifacts or rearrangements and should be provided in an addressable format with clones physically separated. Libraries arrayed in microtiter dishes provide the opportunity for many researchers around the world to accumulate and use information on particular clones (Green and Olson 1990; Nizetic et al. 1991; Evans et al. 1992; Cohen et al. 1993; Marra et al. 1997; Zhao et al. 2000), thus permitting resource sharing through central repositories. BAC libraries are used as a source of substrates for shotgun sequencing projects, to create a database of end sequences (Mahairas et al. 1999; Zhao 2000; Zhao et al. 2000) and restriction fingerprints for building overlapping clone sets (contigs; Marra et al. 1997, 1999). BACs also provide scaffolding information for mapping sequence contigs to localized genomic regions by using a direct genomic shotgun sequencing approach (Adams et al. 2000; Hoskins et al. 2000). The BAC library (RPCI-11) described in this manuscript represents one of the first libraries constructed in compliance with the policies of the U.S. funding agencies for DNA sequence resources and has been used to a larger extent than any other library. A detailed characterization of the RPCI-11 BAC library has not been reported, although its use in clone end sequencing, fingerprinting, and complete Chromosome sequencing has been described (Dunham et al. 1999; Mahairas et al. 1999; Hattori et al. 2000; Soderlund et al. 2000; Zhao et al. 2000; McPherson et al. 2001). To definitively determine that clones contain a single cloned fragment and thus are nonchimeric, both ends of the BAC inserts were independently mapped by screening against other clones within a very redundant clone contig spanning 1.5 Mb of human Chromosome 14. The rationale is that if the BAC is chimeric, the ends will be from different genomic regions. To examine the level of rearrangements within clones, the contig was interrogated with ∼300 markers for inconsistent map results. In addition, all the BACs in the contig also were fingerprinted using high-resolution restriction fragment pattern analysis. This article establishes that chimeric clones containing multiple unrelated genomic DNA segments are essentially absent (at or below the 1% level) and that minor rearrangements can occur in ∼10% of the clones under normal growth conditions.
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bacterial Artificial Chromosome libraries for mouse sequencing and functional analysis
Genome Research, 2000Co-Authors: Kazutoyo Osoegawa, Peng Yeong Woon, Eirik Frengen, Minako Tateno, Joseph J. Catanese, Aaron G MammoserAbstract:Bacterial Artificial Chromosome (BAC) and P1-Derived Artificial Chromosome (PAC) libraries providing a combined 33-fold representation of the murine genome have been constructed using two different restriction enzymes for genomic digestion. A large-insert PAC library was prepared from the 129S6/SvEvTac strain in a bacterial/mammalian shuttle vector to facilitate functional gene studies. For genome mapping and sequencing, we prepared BAC libraries from the 129S6/SvEvTac and the C57BL/6J strains. The average insert sizes for the three libraries range between 130 kb and 200 kb. Based on the numbers of clones and the observed average insert sizes, we estimate each library to have slightly in excess of 10-fold genome representation. The average number of clones found after hybridization screening with 28 probes was in the range of 9-14 clones per marker. To explore the fidelity of the genomic representation in the three libraries, we analyzed three contigs, each established after screening with a single unique marker. New markers were established from the end sequences and screened against all the contig members to determine if any of the BACs and PACs are chimeric or rearranged. Only one chimeric clone and six potential deletions have been observed after extensive analysis of 113 PAC and BAC clones. Seventy-one of the 113 clones were conclusively nonchimeric because both end markers or sequences were mapped to the other confirmed contig members. We could not exclude chimerism for the remaining 41 clones because one or both of the insert termini did not contain unique sequence to design markers. The low rate of chimerism, approximately 1%, and the low level of detected rearrangements support the anticipated usefulness of the BAC libraries for genome research.
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construction and characterization of a 10 fold genome equivalent rat p1 derived Artificial Chromosome library
Genomics, 1998Co-Authors: Kazutoyo Osoegawa, Peng Yeong Woon, Baohui Zhao, Joseph J. Catanese, Pamela J Kaisaki, Dominique Gauguier, Roger D Cox, Elaine R Levy, Mark G LathropAbstract:A rat PAC library was constructed in the vector pPAC4 from genomic DNA isolated from female Brown Norway rats. This library consists of 215,409 clones arrayed in 614 384-well microtiter plates. An average insert size of 143 kb was estimated from 217 randomly isolated clones, thus representing approximately 10-fold genome coverage. This coverage provides a very high probability that the library contains a unique sequence in genome screening. Tests on randomly selected clones demonstrated that they are very stable, with only 4 of 130 clones showing restriction digest fragment alterations after 80 generations of serial growth. FISH analysis using 70 randomly chosen PACs revealed no significant chimeric clones. About 7% of the clones analyzed contained repetitive sequences related to centromeric regions that hybridized to some but not all centromeres. DNA plate pools and superpools were made, and high-density filters each containing an array of 8 plates in duplicate were prepared. Library screening on these superpools and appropriate filters with 10 single-locus rat markers revealed an average of 8 positive clones, in agreement with the estimated high genomic coverage of this library and representation of the rat genome. This library provides a new resource for rat genome analysis, in particular the identification of genes involved in models of multifactorial disease. The library and high-density filters are currently available to the scientific community.
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An Improved Approach for Construction of Bacterial Artificial Chromosome Libraries
Genomics, 1998Co-Authors: Kazutoyo Osoegawa, Peng Yeong Woon, Baohui Zhao, Eirik Frengen, Minako Tateno, Joseph J. Catanese, Pieter J. De JongAbstract:Presented here are improved methodologies that enable the generation of highly redundant bacterial Artificial Chromosome/P1-Derived Artificial Chromosome libraries, with larger and relatively uniform insert sizes. Improvements in vector preparation and enhanced ligation conditions reduce the number of background nonrecombinant clones. Preelectrophoresis of immobilized high-molecular-weight DNA removes inhibitors of the cloning process, while sizing DNA fragments twice within a single gel effectively eliminates small restriction fragments, thus increasing the average insert size of the clones. The size-fractionated DNA fragments are recovered by electroelution rather than the more common melting of gel slices with subsequent β-agarase treatment. Concentration of the ligation products yields a 6- to 12-fold reduction in the number of electroporations required in preparing a library of desirable size. These improved methods have been applied to prepare PAC and BAC libraries from the human, murine, rat, canine, and baboon genomes with average insert sizes ranging between 160 and 235 kb.
Kazutoyo Osoegawa - One of the best experts on this subject based on the ideXlab platform.
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a bacterial Artificial Chromosome library for sequencing the complete human genome
Genome Research, 2001Co-Authors: Kazutoyo Osoegawa, Eirik Frengen, Joseph J. Catanese, Pieter J. De Jong, Aaron G Mammoser, Changjiang ZengAbstract:The main goal of the publicly funded human genome project is to completely determine the human genomic DNA sequence. Five large centers in the United States and the United Kingdom (the G5 group) along with three smaller centers in France, Germany, and Japan (the G8 group) are the major contributors to the sequencing effort. The initial draft version of the human DNA sequence was completed on June 26, 2000, and a high-quality version will become accessible by 2003. The human genome project presents unique ethical and political requirements with respect to the source DNA for library construction, because never before has an individual's genetic blueprint been deciphered completely. One or more volunteers were required to donate their DNA for the sequencing effort. Donor recruitment must comply with regulations (Botkin and Gut 1996; Marshall 1996) to protect the individual's interests and requires informed consent. In addition, it is preferable to obtain the first human genome sequence with the focus on the composition of genes across the prototypical human genome rather than exploring the diversity of genes across the human population. With only a few donors contributing to the prototype of the human genome, it is likely that the prototype will not be equally derived from all ethnic or social groups. To avoid a willful bias with respect to representatives from one group or another, a double-blind donor selection protocol was desirable and was formulated in compliance with the stated policies of the funding agencies (see http://www.nhgri.nih.gov:80/Grant_info/Funding/Statements/RFA/human_subjects.html). Large-insert genomic DNA libraries in bacteria, such as bacterial Artificial Chromosome (BAC; Shizuya et al. 1992) and P1-Derived Artificial Chromosome (PAC; Ioannou et al. 1994) libraries, provide a way to divide the complexity of the human genome into a composite of large DNA segments of reduced complexity. Ideally, BAC libraries should completely represent the genome without cloning artifacts or rearrangements and should be provided in an addressable format with clones physically separated. Libraries arrayed in microtiter dishes provide the opportunity for many researchers around the world to accumulate and use information on particular clones (Green and Olson 1990; Nizetic et al. 1991; Evans et al. 1992; Cohen et al. 1993; Marra et al. 1997; Zhao et al. 2000), thus permitting resource sharing through central repositories. BAC libraries are used as a source of substrates for shotgun sequencing projects, to create a database of end sequences (Mahairas et al. 1999; Zhao 2000; Zhao et al. 2000) and restriction fingerprints for building overlapping clone sets (contigs; Marra et al. 1997, 1999). BACs also provide scaffolding information for mapping sequence contigs to localized genomic regions by using a direct genomic shotgun sequencing approach (Adams et al. 2000; Hoskins et al. 2000). The BAC library (RPCI-11) described in this manuscript represents one of the first libraries constructed in compliance with the policies of the U.S. funding agencies for DNA sequence resources and has been used to a larger extent than any other library. A detailed characterization of the RPCI-11 BAC library has not been reported, although its use in clone end sequencing, fingerprinting, and complete Chromosome sequencing has been described (Dunham et al. 1999; Mahairas et al. 1999; Hattori et al. 2000; Soderlund et al. 2000; Zhao et al. 2000; McPherson et al. 2001). To definitively determine that clones contain a single cloned fragment and thus are nonchimeric, both ends of the BAC inserts were independently mapped by screening against other clones within a very redundant clone contig spanning 1.5 Mb of human Chromosome 14. The rationale is that if the BAC is chimeric, the ends will be from different genomic regions. To examine the level of rearrangements within clones, the contig was interrogated with ∼300 markers for inconsistent map results. In addition, all the BACs in the contig also were fingerprinted using high-resolution restriction fragment pattern analysis. This article establishes that chimeric clones containing multiple unrelated genomic DNA segments are essentially absent (at or below the 1% level) and that minor rearrangements can occur in ∼10% of the clones under normal growth conditions.
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bacterial Artificial Chromosome libraries for mouse sequencing and functional analysis
Genome Research, 2000Co-Authors: Kazutoyo Osoegawa, Peng Yeong Woon, Eirik Frengen, Minako Tateno, Joseph J. Catanese, Aaron G MammoserAbstract:Bacterial Artificial Chromosome (BAC) and P1-Derived Artificial Chromosome (PAC) libraries providing a combined 33-fold representation of the murine genome have been constructed using two different restriction enzymes for genomic digestion. A large-insert PAC library was prepared from the 129S6/SvEvTac strain in a bacterial/mammalian shuttle vector to facilitate functional gene studies. For genome mapping and sequencing, we prepared BAC libraries from the 129S6/SvEvTac and the C57BL/6J strains. The average insert sizes for the three libraries range between 130 kb and 200 kb. Based on the numbers of clones and the observed average insert sizes, we estimate each library to have slightly in excess of 10-fold genome representation. The average number of clones found after hybridization screening with 28 probes was in the range of 9-14 clones per marker. To explore the fidelity of the genomic representation in the three libraries, we analyzed three contigs, each established after screening with a single unique marker. New markers were established from the end sequences and screened against all the contig members to determine if any of the BACs and PACs are chimeric or rearranged. Only one chimeric clone and six potential deletions have been observed after extensive analysis of 113 PAC and BAC clones. Seventy-one of the 113 clones were conclusively nonchimeric because both end markers or sequences were mapped to the other confirmed contig members. We could not exclude chimerism for the remaining 41 clones because one or both of the insert termini did not contain unique sequence to design markers. The low rate of chimerism, approximately 1%, and the low level of detected rearrangements support the anticipated usefulness of the BAC libraries for genome research.
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construction and characterization of a 10 fold genome equivalent rat p1 derived Artificial Chromosome library
Genomics, 1998Co-Authors: Kazutoyo Osoegawa, Peng Yeong Woon, Baohui Zhao, Joseph J. Catanese, Pamela J Kaisaki, Dominique Gauguier, Roger D Cox, Elaine R Levy, Mark G LathropAbstract:A rat PAC library was constructed in the vector pPAC4 from genomic DNA isolated from female Brown Norway rats. This library consists of 215,409 clones arrayed in 614 384-well microtiter plates. An average insert size of 143 kb was estimated from 217 randomly isolated clones, thus representing approximately 10-fold genome coverage. This coverage provides a very high probability that the library contains a unique sequence in genome screening. Tests on randomly selected clones demonstrated that they are very stable, with only 4 of 130 clones showing restriction digest fragment alterations after 80 generations of serial growth. FISH analysis using 70 randomly chosen PACs revealed no significant chimeric clones. About 7% of the clones analyzed contained repetitive sequences related to centromeric regions that hybridized to some but not all centromeres. DNA plate pools and superpools were made, and high-density filters each containing an array of 8 plates in duplicate were prepared. Library screening on these superpools and appropriate filters with 10 single-locus rat markers revealed an average of 8 positive clones, in agreement with the estimated high genomic coverage of this library and representation of the rat genome. This library provides a new resource for rat genome analysis, in particular the identification of genes involved in models of multifactorial disease. The library and high-density filters are currently available to the scientific community.
-
An Improved Approach for Construction of Bacterial Artificial Chromosome Libraries
Genomics, 1998Co-Authors: Kazutoyo Osoegawa, Peng Yeong Woon, Baohui Zhao, Eirik Frengen, Minako Tateno, Joseph J. Catanese, Pieter J. De JongAbstract:Presented here are improved methodologies that enable the generation of highly redundant bacterial Artificial Chromosome/P1-Derived Artificial Chromosome libraries, with larger and relatively uniform insert sizes. Improvements in vector preparation and enhanced ligation conditions reduce the number of background nonrecombinant clones. Preelectrophoresis of immobilized high-molecular-weight DNA removes inhibitors of the cloning process, while sizing DNA fragments twice within a single gel effectively eliminates small restriction fragments, thus increasing the average insert size of the clones. The size-fractionated DNA fragments are recovered by electroelution rather than the more common melting of gel slices with subsequent β-agarase treatment. Concentration of the ligation products yields a 6- to 12-fold reduction in the number of electroporations required in preparing a library of desirable size. These improved methods have been applied to prepare PAC and BAC libraries from the human, murine, rat, canine, and baboon genomes with average insert sizes ranging between 160 and 235 kb.
Adam C. Jones - One of the best experts on this subject based on the ideXlab platform.
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phage p1 derived Artificial Chromosomes facilitate heterologous expression of the fk506 gene cluster
PLOS ONE, 2013Co-Authors: Adam C. Jones, Andreas Kulik, Bertolt Gust, Lutz Heide, Mark J. ButtnerAbstract:We describe a procedure for the conjugative transfer of phage P1-Derived Artificial Chromosome (PAC) library clones containing large natural product gene clusters (≥70 kilobases) to Streptomyces coelicolor strains that have been engineered for improved heterologous production of natural products. This approach is demonstrated using the gene cluster for FK506 (tacrolimus), a clinically important immunosuppressant of high commercial value. The entire 83.5 kb FK506 gene cluster from Streptomyces tsukubaensis NRRL 18488 present in one 130 kb PAC clone was introduced into four different S. coelicolor derivatives and all produced FK506 and smaller amounts of the related compound FK520. FK506 yields were increased by approximately five-fold (from 1.2 mg L-1 to 5.5 mg L-1) in S. coelicolor M1146 containing the FK506 PAC upon over-expression of the FK506 LuxR regulatory gene fkbN. The PAC-based gene cluster conjugation methodology described here provides a tractable means to evaluate and manipulate FK506 biosynthesis and is readily applicable to other large gene clusters encoding natural products of interest to medicine, agriculture and biotechnology.
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Phage P1-Derived Artificial Chromosome (PAC)-based approach for heterologous expression of FK506.
2013Co-Authors: Adam C. Jones, Bertolt Gust, Lutz Heide, Mark J. ButtnerAbstract:A) Structures of FK506 and FK520. B) Vector map of PAC pESAC13. C) Region of Streptomyces tsukubaensis NRRL 18488 genome included in PAC20N and used for heterologous expression. Gene colors refer to the following functions [30]: Yellow, allylmalonyl-CoA biosynthesis; light blue, methoxymalonyl-ACP biosynthesis; dark blue, polyketide synthase; green, chorismate hydrolase (starter unit biosynthesis); brown, peptide synthetase; orange, post-PKS modification; purple, FK506 regulation; red, thioesterase; black, other genes.
