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Zhijun Wang - One of the best experts on this subject based on the ideXlab platform.
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cezomycin is activated by calc to its ester form for further biosynthesis steps in the production of Calcimycin in streptomyces chartreusis nrrl 3882
Applied and Environmental Microbiology, 2018Co-Authors: Hao Wu, Jingdan Liang, Xiufen Zhou, Zixin Deng, Jialiang Wang, Weijun Liang, Qiulin Wu, Ian J Bruce, Zhijun WangAbstract:ABSTRACT Calcimycin, N-demethyl Calcimycin, and cezomycin are polyether divalent cation ionophore secondary metabolites produced by Streptomyces chartreusis. A thorough understanding of the organization of their encoding genes, biosynthetic pathway(s), and cation specificities is vitally important for their efficient future production and therapeutic use. So far, this has been lacking, as has information concerning any biosynthetic relationships that may exist between Calcimycin and cezomycin. In this study, we observed that when a Cal− (calB1 mutant) derivative of a Calcimycin-producing strain of S. chartreusis (NRRL 3882) was grown on cezomycin, Calcimycin production was restored. This suggested that Calcimycin synthesis may have resulted from postsynthetic modification of cezomycin rather than from a de novo process through a novel and independent biosynthetic mechanism. Systematic screening of a number of Cal−S. chartreusis mutants lacking the ability to convert cezomycin to Calcimycin allowed the identification of a gene, provisionally named calC, which was involved in the conversion step. Molecular cloning and heterologous expression of the CalC protein along with its purification to homogeneity and negative-staining electron microscopy allowed the determination of its apparent molecular weight, oligomeric forms in solution, and activity. These experiments allowed us to confirm that the protein possessed ATP pyrophosphatase activity and was capable of ligating coenzyme A (CoA) with cezomycin but not 3-hydroxyanthranilic acid. The CalC protein9s apparent Km and kcat for cezomycin were observed to be 190 μM and 3.98 min−1, respectively, and it possessed the oligomeric form in solution. Our results unequivocally show that cezomycin is postsynthetically modified to Calcimycin by the CalC protein through its activation of cezomycin to a CoA ester form. IMPORTANCE Calcimycin is a secondary metabolite divalent cation-ionophore that has been studied in the context of human health. However, detail is lacking with respect to both Calcimycin9s biosynthesis and its biochemical/biophysical properties as well as information regarding its, and its analogues9, divalent cation binding specificities and other activities. Such knowledge would be useful in understanding how Calcimycin and related compounds may be effective in modifying the calcium channel ion flux and might be useful in influencing the homeostasis of magnesium and manganese ions for the cure or control of human and bacterial infectious diseases. The results presented here unequivocally show that CalC protein is essential for the production of Calcimycin, which is essentially a derivative of cezomycin, and allow us to propose a biosynthetic mechanism for Calcimycin9s production.
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recycling of overactivated acyls by a type ii thioesterase during Calcimycin biosynthesis in streptomyces chartreusis nrrl 3882
Applied and Environmental Microbiology, 2018Co-Authors: Jingdan Liang, Lixia Gou, Xiufen Zhou, Zixin Deng, Weijun Liang, Ian J Bruce, Zhijun WangAbstract:Type II thioesterases typically function as editing enzymes removing acyl groups which have been mis-conjugated to acyl carrier proteins during polyketide secondary metabolite biosynthesis as a consequence of biosynthetic errors. Streptomyces chartreusis NRRL 3882 produces the pyrrole polyether ionophoric antibiotic and we have identified the presence of a putative type II thioesterase like sequence, calG, within the biosynthetic gene cluster involved in the antibiotic9s synthesis. However targeted gene mutagenesis experiments in which calG was inactivated in the organism did not lead to a decrease in Calcimycin production but rather reduced the strain9s production of its main biosynthetic precursor, cezomycin. Results from in vitro activity assays of purified, recombinant CalG protein indicated that it was involved in the hydrolysis of cezomycin-CoA, as well as other acyl CoAs, but was not active toward 3-S-N-acetylcysteamine (SNAC - the mimic of the polyketide chain releasing precursor). Further investigation of the enzyme9s activity showed that it possessed a cezomycin-CoA hydrolysis K m of 0.67 mM and a k cat of 17.77 min -1 and was significantly inhibited by the presence of Mn 2+ , and Fe 2+ divalent cations. Interestingly when S. chartreusis NRRL 3882 was cultured in the presence of inorganic nitrite, NaNO 2, it was observed that the production of Calcimycin rather than cezomycin was promoted. Also that the supplementation of S. chartreusis NRRL 3882 growth medium with the divalent cations Ca 2+ , Mg 2+ , Mn 2+ , and Fe 2+ had a similar effect. Taken together these observations suggest that CalG is not responsible for mega-synthase polyketide precursor chain release during the synthesis of Calcimycin nor for retaining the catalytic efficiency of the mega-synthase enzyme complex as is supposed to be the function for type II thioesterases. Rather our results suggest that CalG is a dedicated thioesterase that prevents the accumulation of cezomycin-CoA when intracellular nitrogen is limited, an apparently new and previously unreported function of Type II thioesterases. Importance Type II thioesterases (TEIIs) are generally regarded as being responsible for removing aberrant acyl groups that block polyketide production thereby maintaining the efficiency of the mega-synthase involved in this class of secondary metabolite9s biosynthesis. Specifically this class of enzyme is believed to be involved in editing mis-primed precursors, controlling initial units, providing key intermediates and releasing final synthetic products in the biosynthesis of this class of secondary metabolite. Our results indicate that the putative TEII, CalG, present in the Calcimycin (A23187) producing organism Streptomyces chartreusis NRRL 3882 is not important either for the retention of catalytic efficiency of, or the release of the product compound from, the mega-synthase involved in Calcimycin biosynthesis. Rather the enzyme is involved in regulating/controlling the pool size of the Calcimycin biosynthetic precursor, cezomycin, by hydrolysis of its CoA derivative. This novel function of CalG suggests a possible additional activity for enzymes belonging to the TEII protein family and promotes better understanding of the overall biosynthetic mechanisms involved in the production of this class of secondary metabolite.
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a novel tetr family transcriptional regulator calr3 negatively controls Calcimycin biosynthesis in streptomyces chartreusis nrrl 3882
Frontiers in Microbiology, 2017Co-Authors: Lixia Gou, Tiesheng Han, Xiaoxia Wang, Wenxiu Liu, Zhijun WangAbstract:Calcimycin is a unique ionophoric antibiotic that is widely used in biochemical and pharmaceutical applications, but the genetic basis underlying the regulatory mechanisms of Calcimycin biosynthesis are unclear. Here, we identified the calR3 gene, which encodes a novel TetR family transcriptional regulator and exerts a negative effect on Calcimycin biosynthesis. Disruption of calR3 in Streptomyces chartreusis NRRL 3882 led to significantly increased Calcimycin and its intermediate cezomycin. Gene expression analysis showed that the transcription of calR3 and its adjacent calT gene were dramatically enhanced (30- and 171-fold, respectively) in GLX26 (ΔcalR3) mutants compared with the wild-type strains. Two CalR3-binding sites within the bidirectional calR3-calT promoter region were identified using a DNase I footprinting assay, indicating that CalR3 directly repressed the transcription of its own gene and the calT gene. In vitro electrophoretic mobility shift assays suggested that both Calcimycin and cezomycin can act as CalR3 ligands to induce CalR3 to dissociate from its binding sites. These findings indicate negative feedback for the regulation of CalR3 in Calcimycin biosynthesis and suggest that Calcimycin production can be improved by manipulating its biosynthetic machinery.
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characterization of the n methyltransferase calm involved in Calcimycin biosynthesis by streptomyces chartreusis nrrl 3882
Biochimie, 2013Co-Authors: Lixia Gou, Shuangjun Lin, Jingdan Liang, Jun Yin, Xiufen Zhou, Linquan Bai, Zixin Deng, Zhijun WangAbstract:Calcimycin is a rare divalent cation specific ionophore antibiotic that has many biochemical and pharmaceutical applications. We have recently cloned and sequenced the Streptomyces chartreusis Calcimycin biosynthesis gene cluster as well as identified the genes required for the synthesis of the polyketide backbone of Calcimycin. Additional modifying or decorating enzymes are required to convert the polyketide backbone into the biologically active Calcimycin. Using targeted mutagenesis of Streptomyces we were able to show that calM from the Calcimycin biosynthesis gene cluster is required for Calcimycin production. Inactivating calM by PCR targeting, caused high level accumulation of N-demethyl Calcimycin. CalM in the presence of S-adenosyl-L-methionine converted N-demethyl Calcimycin to Calcimycin in vitro. The enzyme was determined to have a kinetic parameter of Km 276 μM, kcat 1.26 min(-1) and kcat/Km 76.2 M(-1) s(-1). These results proved that CalM is a N-methyltransferase that is required for Calcimycin biosynthesis, and they set the stage for generating much desired novel Calcimycin derivatives by rational genetic and chemical engineering.
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mutasynthesis of pyrrole spiroketal compound using Calcimycin 3 hydroxy anthranilic acid biosynthetic mutant
Applied Microbiology and Biotechnology, 2013Co-Authors: Lixia Gou, Shuangjun Lin, Jingdan Liang, Xiufen Zhou, Zixin Deng, Zhijun WangAbstract:The five-membered aromatic nitrogen heterocyclic pyrrole ring is a building block for a wide variety of natural products. Aiming at generating new pyrrole-containing derivatives as well as to identify new candidates that may be of value in designing new anticancer, antiviral, and/or antimicrobial agents, we employed a strategy on pyrrole-containing compound mutasynthesis using the pyrrole-containing Calcimycin biosynthetic gene cluster. We blocked the biosynthesis of the Calcimycin precursor, 3-hydroxy anthranilic acid, by deletion of calB1-3 and found that two intermediates containing the pyrrole and the spiroketal moiety were accumulated in the culture. We then fed the mutant using the structurally similar compound of 3-hydroxy anthranilic acid. At least four additional new pyrrole spiroketal derivatives were obtained. The structures of the intermediates and the new pyrrole spiroketal derivatives were identified using LC-MS and NMR. One of them shows enhanced antibacterial activity. Our work shows a new way of pyrrole derivative biosynthetic mutasynthesis.
Lixia Gou - One of the best experts on this subject based on the ideXlab platform.
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recycling of overactivated acyls by a type ii thioesterase during Calcimycin biosynthesis in streptomyces chartreusis nrrl 3882
Applied and Environmental Microbiology, 2018Co-Authors: Jingdan Liang, Lixia Gou, Xiufen Zhou, Zixin Deng, Weijun Liang, Ian J Bruce, Zhijun WangAbstract:Type II thioesterases typically function as editing enzymes removing acyl groups which have been mis-conjugated to acyl carrier proteins during polyketide secondary metabolite biosynthesis as a consequence of biosynthetic errors. Streptomyces chartreusis NRRL 3882 produces the pyrrole polyether ionophoric antibiotic and we have identified the presence of a putative type II thioesterase like sequence, calG, within the biosynthetic gene cluster involved in the antibiotic9s synthesis. However targeted gene mutagenesis experiments in which calG was inactivated in the organism did not lead to a decrease in Calcimycin production but rather reduced the strain9s production of its main biosynthetic precursor, cezomycin. Results from in vitro activity assays of purified, recombinant CalG protein indicated that it was involved in the hydrolysis of cezomycin-CoA, as well as other acyl CoAs, but was not active toward 3-S-N-acetylcysteamine (SNAC - the mimic of the polyketide chain releasing precursor). Further investigation of the enzyme9s activity showed that it possessed a cezomycin-CoA hydrolysis K m of 0.67 mM and a k cat of 17.77 min -1 and was significantly inhibited by the presence of Mn 2+ , and Fe 2+ divalent cations. Interestingly when S. chartreusis NRRL 3882 was cultured in the presence of inorganic nitrite, NaNO 2, it was observed that the production of Calcimycin rather than cezomycin was promoted. Also that the supplementation of S. chartreusis NRRL 3882 growth medium with the divalent cations Ca 2+ , Mg 2+ , Mn 2+ , and Fe 2+ had a similar effect. Taken together these observations suggest that CalG is not responsible for mega-synthase polyketide precursor chain release during the synthesis of Calcimycin nor for retaining the catalytic efficiency of the mega-synthase enzyme complex as is supposed to be the function for type II thioesterases. Rather our results suggest that CalG is a dedicated thioesterase that prevents the accumulation of cezomycin-CoA when intracellular nitrogen is limited, an apparently new and previously unreported function of Type II thioesterases. Importance Type II thioesterases (TEIIs) are generally regarded as being responsible for removing aberrant acyl groups that block polyketide production thereby maintaining the efficiency of the mega-synthase involved in this class of secondary metabolite9s biosynthesis. Specifically this class of enzyme is believed to be involved in editing mis-primed precursors, controlling initial units, providing key intermediates and releasing final synthetic products in the biosynthesis of this class of secondary metabolite. Our results indicate that the putative TEII, CalG, present in the Calcimycin (A23187) producing organism Streptomyces chartreusis NRRL 3882 is not important either for the retention of catalytic efficiency of, or the release of the product compound from, the mega-synthase involved in Calcimycin biosynthesis. Rather the enzyme is involved in regulating/controlling the pool size of the Calcimycin biosynthetic precursor, cezomycin, by hydrolysis of its CoA derivative. This novel function of CalG suggests a possible additional activity for enzymes belonging to the TEII protein family and promotes better understanding of the overall biosynthetic mechanisms involved in the production of this class of secondary metabolite.
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a novel tetr family transcriptional regulator calr3 negatively controls Calcimycin biosynthesis in streptomyces chartreusis nrrl 3882
Frontiers in Microbiology, 2017Co-Authors: Lixia Gou, Tiesheng Han, Xiaoxia Wang, Wenxiu Liu, Zhijun WangAbstract:Calcimycin is a unique ionophoric antibiotic that is widely used in biochemical and pharmaceutical applications, but the genetic basis underlying the regulatory mechanisms of Calcimycin biosynthesis are unclear. Here, we identified the calR3 gene, which encodes a novel TetR family transcriptional regulator and exerts a negative effect on Calcimycin biosynthesis. Disruption of calR3 in Streptomyces chartreusis NRRL 3882 led to significantly increased Calcimycin and its intermediate cezomycin. Gene expression analysis showed that the transcription of calR3 and its adjacent calT gene were dramatically enhanced (30- and 171-fold, respectively) in GLX26 (ΔcalR3) mutants compared with the wild-type strains. Two CalR3-binding sites within the bidirectional calR3-calT promoter region were identified using a DNase I footprinting assay, indicating that CalR3 directly repressed the transcription of its own gene and the calT gene. In vitro electrophoretic mobility shift assays suggested that both Calcimycin and cezomycin can act as CalR3 ligands to induce CalR3 to dissociate from its binding sites. These findings indicate negative feedback for the regulation of CalR3 in Calcimycin biosynthesis and suggest that Calcimycin production can be improved by manipulating its biosynthetic machinery.
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characterization of the n methyltransferase calm involved in Calcimycin biosynthesis by streptomyces chartreusis nrrl 3882
Biochimie, 2013Co-Authors: Lixia Gou, Shuangjun Lin, Jingdan Liang, Jun Yin, Xiufen Zhou, Linquan Bai, Zixin Deng, Zhijun WangAbstract:Calcimycin is a rare divalent cation specific ionophore antibiotic that has many biochemical and pharmaceutical applications. We have recently cloned and sequenced the Streptomyces chartreusis Calcimycin biosynthesis gene cluster as well as identified the genes required for the synthesis of the polyketide backbone of Calcimycin. Additional modifying or decorating enzymes are required to convert the polyketide backbone into the biologically active Calcimycin. Using targeted mutagenesis of Streptomyces we were able to show that calM from the Calcimycin biosynthesis gene cluster is required for Calcimycin production. Inactivating calM by PCR targeting, caused high level accumulation of N-demethyl Calcimycin. CalM in the presence of S-adenosyl-L-methionine converted N-demethyl Calcimycin to Calcimycin in vitro. The enzyme was determined to have a kinetic parameter of Km 276 μM, kcat 1.26 min(-1) and kcat/Km 76.2 M(-1) s(-1). These results proved that CalM is a N-methyltransferase that is required for Calcimycin biosynthesis, and they set the stage for generating much desired novel Calcimycin derivatives by rational genetic and chemical engineering.
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mutasynthesis of pyrrole spiroketal compound using Calcimycin 3 hydroxy anthranilic acid biosynthetic mutant
Applied Microbiology and Biotechnology, 2013Co-Authors: Lixia Gou, Shuangjun Lin, Jingdan Liang, Xiufen Zhou, Zixin Deng, Zhijun WangAbstract:The five-membered aromatic nitrogen heterocyclic pyrrole ring is a building block for a wide variety of natural products. Aiming at generating new pyrrole-containing derivatives as well as to identify new candidates that may be of value in designing new anticancer, antiviral, and/or antimicrobial agents, we employed a strategy on pyrrole-containing compound mutasynthesis using the pyrrole-containing Calcimycin biosynthetic gene cluster. We blocked the biosynthesis of the Calcimycin precursor, 3-hydroxy anthranilic acid, by deletion of calB1-3 and found that two intermediates containing the pyrrole and the spiroketal moiety were accumulated in the culture. We then fed the mutant using the structurally similar compound of 3-hydroxy anthranilic acid. At least four additional new pyrrole spiroketal derivatives were obtained. The structures of the intermediates and the new pyrrole spiroketal derivatives were identified using LC-MS and NMR. One of them shows enhanced antibacterial activity. Our work shows a new way of pyrrole derivative biosynthetic mutasynthesis.
Jingdan Liang - One of the best experts on this subject based on the ideXlab platform.
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cezomycin is activated by calc to its ester form for further biosynthesis steps in the production of Calcimycin in streptomyces chartreusis nrrl 3882
Applied and Environmental Microbiology, 2018Co-Authors: Hao Wu, Jingdan Liang, Xiufen Zhou, Zixin Deng, Jialiang Wang, Weijun Liang, Qiulin Wu, Ian J Bruce, Zhijun WangAbstract:ABSTRACT Calcimycin, N-demethyl Calcimycin, and cezomycin are polyether divalent cation ionophore secondary metabolites produced by Streptomyces chartreusis. A thorough understanding of the organization of their encoding genes, biosynthetic pathway(s), and cation specificities is vitally important for their efficient future production and therapeutic use. So far, this has been lacking, as has information concerning any biosynthetic relationships that may exist between Calcimycin and cezomycin. In this study, we observed that when a Cal− (calB1 mutant) derivative of a Calcimycin-producing strain of S. chartreusis (NRRL 3882) was grown on cezomycin, Calcimycin production was restored. This suggested that Calcimycin synthesis may have resulted from postsynthetic modification of cezomycin rather than from a de novo process through a novel and independent biosynthetic mechanism. Systematic screening of a number of Cal−S. chartreusis mutants lacking the ability to convert cezomycin to Calcimycin allowed the identification of a gene, provisionally named calC, which was involved in the conversion step. Molecular cloning and heterologous expression of the CalC protein along with its purification to homogeneity and negative-staining electron microscopy allowed the determination of its apparent molecular weight, oligomeric forms in solution, and activity. These experiments allowed us to confirm that the protein possessed ATP pyrophosphatase activity and was capable of ligating coenzyme A (CoA) with cezomycin but not 3-hydroxyanthranilic acid. The CalC protein9s apparent Km and kcat for cezomycin were observed to be 190 μM and 3.98 min−1, respectively, and it possessed the oligomeric form in solution. Our results unequivocally show that cezomycin is postsynthetically modified to Calcimycin by the CalC protein through its activation of cezomycin to a CoA ester form. IMPORTANCE Calcimycin is a secondary metabolite divalent cation-ionophore that has been studied in the context of human health. However, detail is lacking with respect to both Calcimycin9s biosynthesis and its biochemical/biophysical properties as well as information regarding its, and its analogues9, divalent cation binding specificities and other activities. Such knowledge would be useful in understanding how Calcimycin and related compounds may be effective in modifying the calcium channel ion flux and might be useful in influencing the homeostasis of magnesium and manganese ions for the cure or control of human and bacterial infectious diseases. The results presented here unequivocally show that CalC protein is essential for the production of Calcimycin, which is essentially a derivative of cezomycin, and allow us to propose a biosynthetic mechanism for Calcimycin9s production.
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recycling of overactivated acyls by a type ii thioesterase during Calcimycin biosynthesis in streptomyces chartreusis nrrl 3882
Applied and Environmental Microbiology, 2018Co-Authors: Jingdan Liang, Lixia Gou, Xiufen Zhou, Zixin Deng, Weijun Liang, Ian J Bruce, Zhijun WangAbstract:Type II thioesterases typically function as editing enzymes removing acyl groups which have been mis-conjugated to acyl carrier proteins during polyketide secondary metabolite biosynthesis as a consequence of biosynthetic errors. Streptomyces chartreusis NRRL 3882 produces the pyrrole polyether ionophoric antibiotic and we have identified the presence of a putative type II thioesterase like sequence, calG, within the biosynthetic gene cluster involved in the antibiotic9s synthesis. However targeted gene mutagenesis experiments in which calG was inactivated in the organism did not lead to a decrease in Calcimycin production but rather reduced the strain9s production of its main biosynthetic precursor, cezomycin. Results from in vitro activity assays of purified, recombinant CalG protein indicated that it was involved in the hydrolysis of cezomycin-CoA, as well as other acyl CoAs, but was not active toward 3-S-N-acetylcysteamine (SNAC - the mimic of the polyketide chain releasing precursor). Further investigation of the enzyme9s activity showed that it possessed a cezomycin-CoA hydrolysis K m of 0.67 mM and a k cat of 17.77 min -1 and was significantly inhibited by the presence of Mn 2+ , and Fe 2+ divalent cations. Interestingly when S. chartreusis NRRL 3882 was cultured in the presence of inorganic nitrite, NaNO 2, it was observed that the production of Calcimycin rather than cezomycin was promoted. Also that the supplementation of S. chartreusis NRRL 3882 growth medium with the divalent cations Ca 2+ , Mg 2+ , Mn 2+ , and Fe 2+ had a similar effect. Taken together these observations suggest that CalG is not responsible for mega-synthase polyketide precursor chain release during the synthesis of Calcimycin nor for retaining the catalytic efficiency of the mega-synthase enzyme complex as is supposed to be the function for type II thioesterases. Rather our results suggest that CalG is a dedicated thioesterase that prevents the accumulation of cezomycin-CoA when intracellular nitrogen is limited, an apparently new and previously unreported function of Type II thioesterases. Importance Type II thioesterases (TEIIs) are generally regarded as being responsible for removing aberrant acyl groups that block polyketide production thereby maintaining the efficiency of the mega-synthase involved in this class of secondary metabolite9s biosynthesis. Specifically this class of enzyme is believed to be involved in editing mis-primed precursors, controlling initial units, providing key intermediates and releasing final synthetic products in the biosynthesis of this class of secondary metabolite. Our results indicate that the putative TEII, CalG, present in the Calcimycin (A23187) producing organism Streptomyces chartreusis NRRL 3882 is not important either for the retention of catalytic efficiency of, or the release of the product compound from, the mega-synthase involved in Calcimycin biosynthesis. Rather the enzyme is involved in regulating/controlling the pool size of the Calcimycin biosynthetic precursor, cezomycin, by hydrolysis of its CoA derivative. This novel function of CalG suggests a possible additional activity for enzymes belonging to the TEII protein family and promotes better understanding of the overall biosynthetic mechanisms involved in the production of this class of secondary metabolite.
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characterization of the n methyltransferase calm involved in Calcimycin biosynthesis by streptomyces chartreusis nrrl 3882
Biochimie, 2013Co-Authors: Lixia Gou, Shuangjun Lin, Jingdan Liang, Jun Yin, Xiufen Zhou, Linquan Bai, Zixin Deng, Zhijun WangAbstract:Calcimycin is a rare divalent cation specific ionophore antibiotic that has many biochemical and pharmaceutical applications. We have recently cloned and sequenced the Streptomyces chartreusis Calcimycin biosynthesis gene cluster as well as identified the genes required for the synthesis of the polyketide backbone of Calcimycin. Additional modifying or decorating enzymes are required to convert the polyketide backbone into the biologically active Calcimycin. Using targeted mutagenesis of Streptomyces we were able to show that calM from the Calcimycin biosynthesis gene cluster is required for Calcimycin production. Inactivating calM by PCR targeting, caused high level accumulation of N-demethyl Calcimycin. CalM in the presence of S-adenosyl-L-methionine converted N-demethyl Calcimycin to Calcimycin in vitro. The enzyme was determined to have a kinetic parameter of Km 276 μM, kcat 1.26 min(-1) and kcat/Km 76.2 M(-1) s(-1). These results proved that CalM is a N-methyltransferase that is required for Calcimycin biosynthesis, and they set the stage for generating much desired novel Calcimycin derivatives by rational genetic and chemical engineering.
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mutasynthesis of pyrrole spiroketal compound using Calcimycin 3 hydroxy anthranilic acid biosynthetic mutant
Applied Microbiology and Biotechnology, 2013Co-Authors: Lixia Gou, Shuangjun Lin, Jingdan Liang, Xiufen Zhou, Zixin Deng, Zhijun WangAbstract:The five-membered aromatic nitrogen heterocyclic pyrrole ring is a building block for a wide variety of natural products. Aiming at generating new pyrrole-containing derivatives as well as to identify new candidates that may be of value in designing new anticancer, antiviral, and/or antimicrobial agents, we employed a strategy on pyrrole-containing compound mutasynthesis using the pyrrole-containing Calcimycin biosynthetic gene cluster. We blocked the biosynthesis of the Calcimycin precursor, 3-hydroxy anthranilic acid, by deletion of calB1-3 and found that two intermediates containing the pyrrole and the spiroketal moiety were accumulated in the culture. We then fed the mutant using the structurally similar compound of 3-hydroxy anthranilic acid. At least four additional new pyrrole spiroketal derivatives were obtained. The structures of the intermediates and the new pyrrole spiroketal derivatives were identified using LC-MS and NMR. One of them shows enhanced antibacterial activity. Our work shows a new way of pyrrole derivative biosynthetic mutasynthesis.
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characterization of the biosynthesis gene cluster for the pyrrole polyether antibiotic Calcimycin a23187 in streptomyces chartreusis nrrl 3882
Antimicrobial Agents and Chemotherapy, 2011Co-Authors: Jingdan Liang, Shuangjun Lin, Xiufen Zhou, Linquan Bai, Zixin Deng, Zhijun WangAbstract:The pyrrole polyether antibiotic Calcimycin (A23187) is a rare ionophore that is specific for divalent cations. It is widely used as a biochemical and pharmacological tool because of its multiple, unique biological effects. Here we report on the cloning, sequencing, and mutational analysis of the 64-kb biosynthetic gene cluster from Streptomyces chartreusis NRRL 3882. Gene replacements confirmed the identity of the gene cluster, and in silico analysis of the DNA sequence revealed 27 potential genes, including 3 genes for the biosynthesis of the α-ketopyrrole moiety, 5 genes that encode modular type I polyketide synthases for the biosynthesis of the spiroketal ring, 4 genes for the biosynthesis of 3-hydroxyanthranilic acid, an N-methyltransferase tailoring gene, a resistance gene, a type II thioesterase gene, 3 regulatory genes, 4 genes with other functions, and 5 genes of unknown function. We propose a pathway for the biosynthesis of Calcimycin and assign the genes to the biosynthesis steps. Our findings set the stage for producing much desired Calcimycin derivatives using genetic modification instead of chemical synthesis.
Zixin Deng - One of the best experts on this subject based on the ideXlab platform.
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cezomycin is activated by calc to its ester form for further biosynthesis steps in the production of Calcimycin in streptomyces chartreusis nrrl 3882
Applied and Environmental Microbiology, 2018Co-Authors: Hao Wu, Jingdan Liang, Xiufen Zhou, Zixin Deng, Jialiang Wang, Weijun Liang, Qiulin Wu, Ian J Bruce, Zhijun WangAbstract:ABSTRACT Calcimycin, N-demethyl Calcimycin, and cezomycin are polyether divalent cation ionophore secondary metabolites produced by Streptomyces chartreusis. A thorough understanding of the organization of their encoding genes, biosynthetic pathway(s), and cation specificities is vitally important for their efficient future production and therapeutic use. So far, this has been lacking, as has information concerning any biosynthetic relationships that may exist between Calcimycin and cezomycin. In this study, we observed that when a Cal− (calB1 mutant) derivative of a Calcimycin-producing strain of S. chartreusis (NRRL 3882) was grown on cezomycin, Calcimycin production was restored. This suggested that Calcimycin synthesis may have resulted from postsynthetic modification of cezomycin rather than from a de novo process through a novel and independent biosynthetic mechanism. Systematic screening of a number of Cal−S. chartreusis mutants lacking the ability to convert cezomycin to Calcimycin allowed the identification of a gene, provisionally named calC, which was involved in the conversion step. Molecular cloning and heterologous expression of the CalC protein along with its purification to homogeneity and negative-staining electron microscopy allowed the determination of its apparent molecular weight, oligomeric forms in solution, and activity. These experiments allowed us to confirm that the protein possessed ATP pyrophosphatase activity and was capable of ligating coenzyme A (CoA) with cezomycin but not 3-hydroxyanthranilic acid. The CalC protein9s apparent Km and kcat for cezomycin were observed to be 190 μM and 3.98 min−1, respectively, and it possessed the oligomeric form in solution. Our results unequivocally show that cezomycin is postsynthetically modified to Calcimycin by the CalC protein through its activation of cezomycin to a CoA ester form. IMPORTANCE Calcimycin is a secondary metabolite divalent cation-ionophore that has been studied in the context of human health. However, detail is lacking with respect to both Calcimycin9s biosynthesis and its biochemical/biophysical properties as well as information regarding its, and its analogues9, divalent cation binding specificities and other activities. Such knowledge would be useful in understanding how Calcimycin and related compounds may be effective in modifying the calcium channel ion flux and might be useful in influencing the homeostasis of magnesium and manganese ions for the cure or control of human and bacterial infectious diseases. The results presented here unequivocally show that CalC protein is essential for the production of Calcimycin, which is essentially a derivative of cezomycin, and allow us to propose a biosynthetic mechanism for Calcimycin9s production.
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recycling of overactivated acyls by a type ii thioesterase during Calcimycin biosynthesis in streptomyces chartreusis nrrl 3882
Applied and Environmental Microbiology, 2018Co-Authors: Jingdan Liang, Lixia Gou, Xiufen Zhou, Zixin Deng, Weijun Liang, Ian J Bruce, Zhijun WangAbstract:Type II thioesterases typically function as editing enzymes removing acyl groups which have been mis-conjugated to acyl carrier proteins during polyketide secondary metabolite biosynthesis as a consequence of biosynthetic errors. Streptomyces chartreusis NRRL 3882 produces the pyrrole polyether ionophoric antibiotic and we have identified the presence of a putative type II thioesterase like sequence, calG, within the biosynthetic gene cluster involved in the antibiotic9s synthesis. However targeted gene mutagenesis experiments in which calG was inactivated in the organism did not lead to a decrease in Calcimycin production but rather reduced the strain9s production of its main biosynthetic precursor, cezomycin. Results from in vitro activity assays of purified, recombinant CalG protein indicated that it was involved in the hydrolysis of cezomycin-CoA, as well as other acyl CoAs, but was not active toward 3-S-N-acetylcysteamine (SNAC - the mimic of the polyketide chain releasing precursor). Further investigation of the enzyme9s activity showed that it possessed a cezomycin-CoA hydrolysis K m of 0.67 mM and a k cat of 17.77 min -1 and was significantly inhibited by the presence of Mn 2+ , and Fe 2+ divalent cations. Interestingly when S. chartreusis NRRL 3882 was cultured in the presence of inorganic nitrite, NaNO 2, it was observed that the production of Calcimycin rather than cezomycin was promoted. Also that the supplementation of S. chartreusis NRRL 3882 growth medium with the divalent cations Ca 2+ , Mg 2+ , Mn 2+ , and Fe 2+ had a similar effect. Taken together these observations suggest that CalG is not responsible for mega-synthase polyketide precursor chain release during the synthesis of Calcimycin nor for retaining the catalytic efficiency of the mega-synthase enzyme complex as is supposed to be the function for type II thioesterases. Rather our results suggest that CalG is a dedicated thioesterase that prevents the accumulation of cezomycin-CoA when intracellular nitrogen is limited, an apparently new and previously unreported function of Type II thioesterases. Importance Type II thioesterases (TEIIs) are generally regarded as being responsible for removing aberrant acyl groups that block polyketide production thereby maintaining the efficiency of the mega-synthase involved in this class of secondary metabolite9s biosynthesis. Specifically this class of enzyme is believed to be involved in editing mis-primed precursors, controlling initial units, providing key intermediates and releasing final synthetic products in the biosynthesis of this class of secondary metabolite. Our results indicate that the putative TEII, CalG, present in the Calcimycin (A23187) producing organism Streptomyces chartreusis NRRL 3882 is not important either for the retention of catalytic efficiency of, or the release of the product compound from, the mega-synthase involved in Calcimycin biosynthesis. Rather the enzyme is involved in regulating/controlling the pool size of the Calcimycin biosynthetic precursor, cezomycin, by hydrolysis of its CoA derivative. This novel function of CalG suggests a possible additional activity for enzymes belonging to the TEII protein family and promotes better understanding of the overall biosynthetic mechanisms involved in the production of this class of secondary metabolite.
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characterization of the n methyltransferase calm involved in Calcimycin biosynthesis by streptomyces chartreusis nrrl 3882
Biochimie, 2013Co-Authors: Lixia Gou, Shuangjun Lin, Jingdan Liang, Jun Yin, Xiufen Zhou, Linquan Bai, Zixin Deng, Zhijun WangAbstract:Calcimycin is a rare divalent cation specific ionophore antibiotic that has many biochemical and pharmaceutical applications. We have recently cloned and sequenced the Streptomyces chartreusis Calcimycin biosynthesis gene cluster as well as identified the genes required for the synthesis of the polyketide backbone of Calcimycin. Additional modifying or decorating enzymes are required to convert the polyketide backbone into the biologically active Calcimycin. Using targeted mutagenesis of Streptomyces we were able to show that calM from the Calcimycin biosynthesis gene cluster is required for Calcimycin production. Inactivating calM by PCR targeting, caused high level accumulation of N-demethyl Calcimycin. CalM in the presence of S-adenosyl-L-methionine converted N-demethyl Calcimycin to Calcimycin in vitro. The enzyme was determined to have a kinetic parameter of Km 276 μM, kcat 1.26 min(-1) and kcat/Km 76.2 M(-1) s(-1). These results proved that CalM is a N-methyltransferase that is required for Calcimycin biosynthesis, and they set the stage for generating much desired novel Calcimycin derivatives by rational genetic and chemical engineering.
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mutasynthesis of pyrrole spiroketal compound using Calcimycin 3 hydroxy anthranilic acid biosynthetic mutant
Applied Microbiology and Biotechnology, 2013Co-Authors: Lixia Gou, Shuangjun Lin, Jingdan Liang, Xiufen Zhou, Zixin Deng, Zhijun WangAbstract:The five-membered aromatic nitrogen heterocyclic pyrrole ring is a building block for a wide variety of natural products. Aiming at generating new pyrrole-containing derivatives as well as to identify new candidates that may be of value in designing new anticancer, antiviral, and/or antimicrobial agents, we employed a strategy on pyrrole-containing compound mutasynthesis using the pyrrole-containing Calcimycin biosynthetic gene cluster. We blocked the biosynthesis of the Calcimycin precursor, 3-hydroxy anthranilic acid, by deletion of calB1-3 and found that two intermediates containing the pyrrole and the spiroketal moiety were accumulated in the culture. We then fed the mutant using the structurally similar compound of 3-hydroxy anthranilic acid. At least four additional new pyrrole spiroketal derivatives were obtained. The structures of the intermediates and the new pyrrole spiroketal derivatives were identified using LC-MS and NMR. One of them shows enhanced antibacterial activity. Our work shows a new way of pyrrole derivative biosynthetic mutasynthesis.
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characterization of the biosynthesis gene cluster for the pyrrole polyether antibiotic Calcimycin a23187 in streptomyces chartreusis nrrl 3882
Antimicrobial Agents and Chemotherapy, 2011Co-Authors: Jingdan Liang, Shuangjun Lin, Xiufen Zhou, Linquan Bai, Zixin Deng, Zhijun WangAbstract:The pyrrole polyether antibiotic Calcimycin (A23187) is a rare ionophore that is specific for divalent cations. It is widely used as a biochemical and pharmacological tool because of its multiple, unique biological effects. Here we report on the cloning, sequencing, and mutational analysis of the 64-kb biosynthetic gene cluster from Streptomyces chartreusis NRRL 3882. Gene replacements confirmed the identity of the gene cluster, and in silico analysis of the DNA sequence revealed 27 potential genes, including 3 genes for the biosynthesis of the α-ketopyrrole moiety, 5 genes that encode modular type I polyketide synthases for the biosynthesis of the spiroketal ring, 4 genes for the biosynthesis of 3-hydroxyanthranilic acid, an N-methyltransferase tailoring gene, a resistance gene, a type II thioesterase gene, 3 regulatory genes, 4 genes with other functions, and 5 genes of unknown function. We propose a pathway for the biosynthesis of Calcimycin and assign the genes to the biosynthesis steps. Our findings set the stage for producing much desired Calcimycin derivatives using genetic modification instead of chemical synthesis.
Xiufen Zhou - One of the best experts on this subject based on the ideXlab platform.
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cezomycin is activated by calc to its ester form for further biosynthesis steps in the production of Calcimycin in streptomyces chartreusis nrrl 3882
Applied and Environmental Microbiology, 2018Co-Authors: Hao Wu, Jingdan Liang, Xiufen Zhou, Zixin Deng, Jialiang Wang, Weijun Liang, Qiulin Wu, Ian J Bruce, Zhijun WangAbstract:ABSTRACT Calcimycin, N-demethyl Calcimycin, and cezomycin are polyether divalent cation ionophore secondary metabolites produced by Streptomyces chartreusis. A thorough understanding of the organization of their encoding genes, biosynthetic pathway(s), and cation specificities is vitally important for their efficient future production and therapeutic use. So far, this has been lacking, as has information concerning any biosynthetic relationships that may exist between Calcimycin and cezomycin. In this study, we observed that when a Cal− (calB1 mutant) derivative of a Calcimycin-producing strain of S. chartreusis (NRRL 3882) was grown on cezomycin, Calcimycin production was restored. This suggested that Calcimycin synthesis may have resulted from postsynthetic modification of cezomycin rather than from a de novo process through a novel and independent biosynthetic mechanism. Systematic screening of a number of Cal−S. chartreusis mutants lacking the ability to convert cezomycin to Calcimycin allowed the identification of a gene, provisionally named calC, which was involved in the conversion step. Molecular cloning and heterologous expression of the CalC protein along with its purification to homogeneity and negative-staining electron microscopy allowed the determination of its apparent molecular weight, oligomeric forms in solution, and activity. These experiments allowed us to confirm that the protein possessed ATP pyrophosphatase activity and was capable of ligating coenzyme A (CoA) with cezomycin but not 3-hydroxyanthranilic acid. The CalC protein9s apparent Km and kcat for cezomycin were observed to be 190 μM and 3.98 min−1, respectively, and it possessed the oligomeric form in solution. Our results unequivocally show that cezomycin is postsynthetically modified to Calcimycin by the CalC protein through its activation of cezomycin to a CoA ester form. IMPORTANCE Calcimycin is a secondary metabolite divalent cation-ionophore that has been studied in the context of human health. However, detail is lacking with respect to both Calcimycin9s biosynthesis and its biochemical/biophysical properties as well as information regarding its, and its analogues9, divalent cation binding specificities and other activities. Such knowledge would be useful in understanding how Calcimycin and related compounds may be effective in modifying the calcium channel ion flux and might be useful in influencing the homeostasis of magnesium and manganese ions for the cure or control of human and bacterial infectious diseases. The results presented here unequivocally show that CalC protein is essential for the production of Calcimycin, which is essentially a derivative of cezomycin, and allow us to propose a biosynthetic mechanism for Calcimycin9s production.
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recycling of overactivated acyls by a type ii thioesterase during Calcimycin biosynthesis in streptomyces chartreusis nrrl 3882
Applied and Environmental Microbiology, 2018Co-Authors: Jingdan Liang, Lixia Gou, Xiufen Zhou, Zixin Deng, Weijun Liang, Ian J Bruce, Zhijun WangAbstract:Type II thioesterases typically function as editing enzymes removing acyl groups which have been mis-conjugated to acyl carrier proteins during polyketide secondary metabolite biosynthesis as a consequence of biosynthetic errors. Streptomyces chartreusis NRRL 3882 produces the pyrrole polyether ionophoric antibiotic and we have identified the presence of a putative type II thioesterase like sequence, calG, within the biosynthetic gene cluster involved in the antibiotic9s synthesis. However targeted gene mutagenesis experiments in which calG was inactivated in the organism did not lead to a decrease in Calcimycin production but rather reduced the strain9s production of its main biosynthetic precursor, cezomycin. Results from in vitro activity assays of purified, recombinant CalG protein indicated that it was involved in the hydrolysis of cezomycin-CoA, as well as other acyl CoAs, but was not active toward 3-S-N-acetylcysteamine (SNAC - the mimic of the polyketide chain releasing precursor). Further investigation of the enzyme9s activity showed that it possessed a cezomycin-CoA hydrolysis K m of 0.67 mM and a k cat of 17.77 min -1 and was significantly inhibited by the presence of Mn 2+ , and Fe 2+ divalent cations. Interestingly when S. chartreusis NRRL 3882 was cultured in the presence of inorganic nitrite, NaNO 2, it was observed that the production of Calcimycin rather than cezomycin was promoted. Also that the supplementation of S. chartreusis NRRL 3882 growth medium with the divalent cations Ca 2+ , Mg 2+ , Mn 2+ , and Fe 2+ had a similar effect. Taken together these observations suggest that CalG is not responsible for mega-synthase polyketide precursor chain release during the synthesis of Calcimycin nor for retaining the catalytic efficiency of the mega-synthase enzyme complex as is supposed to be the function for type II thioesterases. Rather our results suggest that CalG is a dedicated thioesterase that prevents the accumulation of cezomycin-CoA when intracellular nitrogen is limited, an apparently new and previously unreported function of Type II thioesterases. Importance Type II thioesterases (TEIIs) are generally regarded as being responsible for removing aberrant acyl groups that block polyketide production thereby maintaining the efficiency of the mega-synthase involved in this class of secondary metabolite9s biosynthesis. Specifically this class of enzyme is believed to be involved in editing mis-primed precursors, controlling initial units, providing key intermediates and releasing final synthetic products in the biosynthesis of this class of secondary metabolite. Our results indicate that the putative TEII, CalG, present in the Calcimycin (A23187) producing organism Streptomyces chartreusis NRRL 3882 is not important either for the retention of catalytic efficiency of, or the release of the product compound from, the mega-synthase involved in Calcimycin biosynthesis. Rather the enzyme is involved in regulating/controlling the pool size of the Calcimycin biosynthetic precursor, cezomycin, by hydrolysis of its CoA derivative. This novel function of CalG suggests a possible additional activity for enzymes belonging to the TEII protein family and promotes better understanding of the overall biosynthetic mechanisms involved in the production of this class of secondary metabolite.
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characterization of the n methyltransferase calm involved in Calcimycin biosynthesis by streptomyces chartreusis nrrl 3882
Biochimie, 2013Co-Authors: Lixia Gou, Shuangjun Lin, Jingdan Liang, Jun Yin, Xiufen Zhou, Linquan Bai, Zixin Deng, Zhijun WangAbstract:Calcimycin is a rare divalent cation specific ionophore antibiotic that has many biochemical and pharmaceutical applications. We have recently cloned and sequenced the Streptomyces chartreusis Calcimycin biosynthesis gene cluster as well as identified the genes required for the synthesis of the polyketide backbone of Calcimycin. Additional modifying or decorating enzymes are required to convert the polyketide backbone into the biologically active Calcimycin. Using targeted mutagenesis of Streptomyces we were able to show that calM from the Calcimycin biosynthesis gene cluster is required for Calcimycin production. Inactivating calM by PCR targeting, caused high level accumulation of N-demethyl Calcimycin. CalM in the presence of S-adenosyl-L-methionine converted N-demethyl Calcimycin to Calcimycin in vitro. The enzyme was determined to have a kinetic parameter of Km 276 μM, kcat 1.26 min(-1) and kcat/Km 76.2 M(-1) s(-1). These results proved that CalM is a N-methyltransferase that is required for Calcimycin biosynthesis, and they set the stage for generating much desired novel Calcimycin derivatives by rational genetic and chemical engineering.
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mutasynthesis of pyrrole spiroketal compound using Calcimycin 3 hydroxy anthranilic acid biosynthetic mutant
Applied Microbiology and Biotechnology, 2013Co-Authors: Lixia Gou, Shuangjun Lin, Jingdan Liang, Xiufen Zhou, Zixin Deng, Zhijun WangAbstract:The five-membered aromatic nitrogen heterocyclic pyrrole ring is a building block for a wide variety of natural products. Aiming at generating new pyrrole-containing derivatives as well as to identify new candidates that may be of value in designing new anticancer, antiviral, and/or antimicrobial agents, we employed a strategy on pyrrole-containing compound mutasynthesis using the pyrrole-containing Calcimycin biosynthetic gene cluster. We blocked the biosynthesis of the Calcimycin precursor, 3-hydroxy anthranilic acid, by deletion of calB1-3 and found that two intermediates containing the pyrrole and the spiroketal moiety were accumulated in the culture. We then fed the mutant using the structurally similar compound of 3-hydroxy anthranilic acid. At least four additional new pyrrole spiroketal derivatives were obtained. The structures of the intermediates and the new pyrrole spiroketal derivatives were identified using LC-MS and NMR. One of them shows enhanced antibacterial activity. Our work shows a new way of pyrrole derivative biosynthetic mutasynthesis.
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characterization of the biosynthesis gene cluster for the pyrrole polyether antibiotic Calcimycin a23187 in streptomyces chartreusis nrrl 3882
Antimicrobial Agents and Chemotherapy, 2011Co-Authors: Jingdan Liang, Shuangjun Lin, Xiufen Zhou, Linquan Bai, Zixin Deng, Zhijun WangAbstract:The pyrrole polyether antibiotic Calcimycin (A23187) is a rare ionophore that is specific for divalent cations. It is widely used as a biochemical and pharmacological tool because of its multiple, unique biological effects. Here we report on the cloning, sequencing, and mutational analysis of the 64-kb biosynthetic gene cluster from Streptomyces chartreusis NRRL 3882. Gene replacements confirmed the identity of the gene cluster, and in silico analysis of the DNA sequence revealed 27 potential genes, including 3 genes for the biosynthesis of the α-ketopyrrole moiety, 5 genes that encode modular type I polyketide synthases for the biosynthesis of the spiroketal ring, 4 genes for the biosynthesis of 3-hydroxyanthranilic acid, an N-methyltransferase tailoring gene, a resistance gene, a type II thioesterase gene, 3 regulatory genes, 4 genes with other functions, and 5 genes of unknown function. We propose a pathway for the biosynthesis of Calcimycin and assign the genes to the biosynthesis steps. Our findings set the stage for producing much desired Calcimycin derivatives using genetic modification instead of chemical synthesis.