Investigations on the early biosynthetic steps of griseorhodin A
Investigations on the early biosynthetic steps of griseorhodin A
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DissertationDate of Exam
16.12.2011Date of Publication
03.05.2012Advisor
Piel, JörnCo-Referee
Sahl, Hans-GeorgMetadata
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Abstract
Presented here are the results concerning the early biosynthesis of the telomerase inhibitor griseorhodin A.
The griseorhodin biosynthetic gene cluster encodes components of a type II polyketide synthase (PKS). The cluster consists of 33 genes, many of which encode oxidoreductases. The grh biosynthetic cluster was isolated from Streptomyces sp. JP95, a bacterium isolated from the marine tunicate Aplidium lenticulum collected in the Great Barrier Reef, Australia. The complete grh cluster is available for expression on the cosmid pMP31, which can be heterologously expressed in S. albus J1074.
To investigate the early biosynthetic steps in grh biosynthesis, the genes grhA, grhB, grhC, grhE, grhT and grhQ were cloned for heterologous expression in the host organism S. albus. S. albus strains expressing the grh minimal PKS with and without the grh cyclases did not result in detectable amounts of polyketides or shunt products. This is in high contrast to other type II PKS systems. In an alternative approach, all cyclase genes were individually deleted withinin the grh cluster. Based on these results, the enzyme GrhE does not seem to play a significant role in the grh biosynthesis since griseorhodin A was still produced. The deletion of the other cyclases, GrhT and GrhQ, abolished the biosynthesis and no polyketides were detected. This suggests that a multienzyme complex is required for early polyketide assembly. To further investigate early griseorhodin A biosynthesis, two other griseorhodin A gene deletion mutants, ΔgrhH-P (deletion of all post-PKS genes) and ΔgrhH-Q (deletion of all post-PKS genes and a cyclase gene), were generated during the present study. The ΔgrhH-P mutant contained only the proposed early griseorhodin genes. Heterologous expression of this construct led to the production of a complex mixture of compounds. It produced identical intermediates to the ΔgrhM mutant, which was generated and expressed during a previous study and for which an early oxygenation role is proposed during griseorhodin A biosynthesis. Four compounds from the ΔgrhH-P mutant expression were isolated and analysed indicating potentially new early intermediates. However, in sufficient material was isolated for structure elucidation by 2D NMR.
To further investigate whether a functional protein complex is relevant at the early stage of griseorhodin A biosynthesis, in the second mutant ΔgrhH-Q, the cyclase GrhQ was additionally deleted, and the expression led to abolishment of griseorhodin A biosynthesis. This was in agreement with the GrhQ inactivation study. These results indicate and further support the presence of a multienzyme complex during the course of griseorhodin A biosynthesis. To date, griseorhodin A is one of the longest aromatic polyketides and therefore, could require the synergistic actions of cyclases (GrhQ) and other stabilising enzymes such as GrhS for correct polyketide assembly.
In order to gain deeper insights into the enzymatic activities responsible for griseorhodin A biosynthesis, two constructs with single gene deletions within the cluster, constructed in a previous study were further investigated. The two deleted genes, grhU and grhV encode proteins with unknown function. In the present study, these modified clusters were subsequently heterologously expressed in S. albus and analysed by high performance liquid chromatography (HPLC), high resolution mass spectroscopy (HRMS) and cryogenic or capillary NMR for the production of secondary metabolites. Heterologous expression of both mutants resulted in complex mixtures of compounds, and 21 pure compounds were isolated by various chromatographic methods. The structure of one of the compounds originating from the ΔgrhV gene deletion mutant was elucidated from 500 μg of sample and the structure revealed the decarboxylated 13,13´- fused tridecaketide dimer. The structure suggests that GrhV is responsible for an oxidative modification in the griseorhodin A molecule. To our knowledge, the present study gives first insights into the oxygenase function of GrhV and its homologues found in other pentangular biosynthetic pathways.
The griseorhodin biosynthetic gene cluster encodes components of a type II polyketide synthase (PKS). The cluster consists of 33 genes, many of which encode oxidoreductases. The grh biosynthetic cluster was isolated from Streptomyces sp. JP95, a bacterium isolated from the marine tunicate Aplidium lenticulum collected in the Great Barrier Reef, Australia. The complete grh cluster is available for expression on the cosmid pMP31, which can be heterologously expressed in S. albus J1074.
To investigate the early biosynthetic steps in grh biosynthesis, the genes grhA, grhB, grhC, grhE, grhT and grhQ were cloned for heterologous expression in the host organism S. albus. S. albus strains expressing the grh minimal PKS with and without the grh cyclases did not result in detectable amounts of polyketides or shunt products. This is in high contrast to other type II PKS systems. In an alternative approach, all cyclase genes were individually deleted withinin the grh cluster. Based on these results, the enzyme GrhE does not seem to play a significant role in the grh biosynthesis since griseorhodin A was still produced. The deletion of the other cyclases, GrhT and GrhQ, abolished the biosynthesis and no polyketides were detected. This suggests that a multienzyme complex is required for early polyketide assembly. To further investigate early griseorhodin A biosynthesis, two other griseorhodin A gene deletion mutants, ΔgrhH-P (deletion of all post-PKS genes) and ΔgrhH-Q (deletion of all post-PKS genes and a cyclase gene), were generated during the present study. The ΔgrhH-P mutant contained only the proposed early griseorhodin genes. Heterologous expression of this construct led to the production of a complex mixture of compounds. It produced identical intermediates to the ΔgrhM mutant, which was generated and expressed during a previous study and for which an early oxygenation role is proposed during griseorhodin A biosynthesis. Four compounds from the ΔgrhH-P mutant expression were isolated and analysed indicating potentially new early intermediates. However, in sufficient material was isolated for structure elucidation by 2D NMR.
To further investigate whether a functional protein complex is relevant at the early stage of griseorhodin A biosynthesis, in the second mutant ΔgrhH-Q, the cyclase GrhQ was additionally deleted, and the expression led to abolishment of griseorhodin A biosynthesis. This was in agreement with the GrhQ inactivation study. These results indicate and further support the presence of a multienzyme complex during the course of griseorhodin A biosynthesis. To date, griseorhodin A is one of the longest aromatic polyketides and therefore, could require the synergistic actions of cyclases (GrhQ) and other stabilising enzymes such as GrhS for correct polyketide assembly.
In order to gain deeper insights into the enzymatic activities responsible for griseorhodin A biosynthesis, two constructs with single gene deletions within the cluster, constructed in a previous study were further investigated. The two deleted genes, grhU and grhV encode proteins with unknown function. In the present study, these modified clusters were subsequently heterologously expressed in S. albus and analysed by high performance liquid chromatography (HPLC), high resolution mass spectroscopy (HRMS) and cryogenic or capillary NMR for the production of secondary metabolites. Heterologous expression of both mutants resulted in complex mixtures of compounds, and 21 pure compounds were isolated by various chromatographic methods. The structure of one of the compounds originating from the ΔgrhV gene deletion mutant was elucidated from 500 μg of sample and the structure revealed the decarboxylated 13,13´- fused tridecaketide dimer. The structure suggests that GrhV is responsible for an oxidative modification in the griseorhodin A molecule. To our knowledge, the present study gives first insights into the oxygenase function of GrhV and its homologues found in other pentangular biosynthetic pathways.
Classification (DDC)
540 Chemie 570 Biowissenschaften, BiologieEklund, Minna Leena: Investigations on the early biosynthetic steps of griseorhodin A. - Bonn, 2012. - Dissertation, Rheinische Friedrich-Wilhelms-Universität Bonn.
Online-Ausgabe in bonndoc: https://nbn-resolving.org/urn:nbn:de:hbz:5n-27901
Online-Ausgabe in bonndoc: https://nbn-resolving.org/urn:nbn:de:hbz:5n-27901
@phdthesis{handle:20.500.11811/5283,
urn: https://nbn-resolving.org/urn:nbn:de:hbz:5n-27901,
author = {{Minna Leena Eklund}},
title = {Investigations on the early biosynthetic steps of griseorhodin A},
school = {Rheinische Friedrich-Wilhelms-Universität Bonn},
year = 2012,
month = may,
note = {Presented here are the results concerning the early biosynthesis of the telomerase inhibitor griseorhodin A.
The griseorhodin biosynthetic gene cluster encodes components of a type II polyketide synthase (PKS). The cluster consists of 33 genes, many of which encode oxidoreductases. The grh biosynthetic cluster was isolated from Streptomyces sp. JP95, a bacterium isolated from the marine tunicate Aplidium lenticulum collected in the Great Barrier Reef, Australia. The complete grh cluster is available for expression on the cosmid pMP31, which can be heterologously expressed in S. albus J1074.
To investigate the early biosynthetic steps in grh biosynthesis, the genes grhA, grhB, grhC, grhE, grhT and grhQ were cloned for heterologous expression in the host organism S. albus. S. albus strains expressing the grh minimal PKS with and without the grh cyclases did not result in detectable amounts of polyketides or shunt products. This is in high contrast to other type II PKS systems. In an alternative approach, all cyclase genes were individually deleted withinin the grh cluster. Based on these results, the enzyme GrhE does not seem to play a significant role in the grh biosynthesis since griseorhodin A was still produced. The deletion of the other cyclases, GrhT and GrhQ, abolished the biosynthesis and no polyketides were detected. This suggests that a multienzyme complex is required for early polyketide assembly. To further investigate early griseorhodin A biosynthesis, two other griseorhodin A gene deletion mutants, ΔgrhH-P (deletion of all post-PKS genes) and ΔgrhH-Q (deletion of all post-PKS genes and a cyclase gene), were generated during the present study. The ΔgrhH-P mutant contained only the proposed early griseorhodin genes. Heterologous expression of this construct led to the production of a complex mixture of compounds. It produced identical intermediates to the ΔgrhM mutant, which was generated and expressed during a previous study and for which an early oxygenation role is proposed during griseorhodin A biosynthesis. Four compounds from the ΔgrhH-P mutant expression were isolated and analysed indicating potentially new early intermediates. However, in sufficient material was isolated for structure elucidation by 2D NMR.
To further investigate whether a functional protein complex is relevant at the early stage of griseorhodin A biosynthesis, in the second mutant ΔgrhH-Q, the cyclase GrhQ was additionally deleted, and the expression led to abolishment of griseorhodin A biosynthesis. This was in agreement with the GrhQ inactivation study. These results indicate and further support the presence of a multienzyme complex during the course of griseorhodin A biosynthesis. To date, griseorhodin A is one of the longest aromatic polyketides and therefore, could require the synergistic actions of cyclases (GrhQ) and other stabilising enzymes such as GrhS for correct polyketide assembly.
In order to gain deeper insights into the enzymatic activities responsible for griseorhodin A biosynthesis, two constructs with single gene deletions within the cluster, constructed in a previous study were further investigated. The two deleted genes, grhU and grhV encode proteins with unknown function. In the present study, these modified clusters were subsequently heterologously expressed in S. albus and analysed by high performance liquid chromatography (HPLC), high resolution mass spectroscopy (HRMS) and cryogenic or capillary NMR for the production of secondary metabolites. Heterologous expression of both mutants resulted in complex mixtures of compounds, and 21 pure compounds were isolated by various chromatographic methods. The structure of one of the compounds originating from the ΔgrhV gene deletion mutant was elucidated from 500 μg of sample and the structure revealed the decarboxylated 13,13´- fused tridecaketide dimer. The structure suggests that GrhV is responsible for an oxidative modification in the griseorhodin A molecule. To our knowledge, the present study gives first insights into the oxygenase function of GrhV and its homologues found in other pentangular biosynthetic pathways.},
url = {https://hdl.handle.net/20.500.11811/5283}
}
urn: https://nbn-resolving.org/urn:nbn:de:hbz:5n-27901,
author = {{Minna Leena Eklund}},
title = {Investigations on the early biosynthetic steps of griseorhodin A},
school = {Rheinische Friedrich-Wilhelms-Universität Bonn},
year = 2012,
month = may,
note = {Presented here are the results concerning the early biosynthesis of the telomerase inhibitor griseorhodin A.
The griseorhodin biosynthetic gene cluster encodes components of a type II polyketide synthase (PKS). The cluster consists of 33 genes, many of which encode oxidoreductases. The grh biosynthetic cluster was isolated from Streptomyces sp. JP95, a bacterium isolated from the marine tunicate Aplidium lenticulum collected in the Great Barrier Reef, Australia. The complete grh cluster is available for expression on the cosmid pMP31, which can be heterologously expressed in S. albus J1074.
To investigate the early biosynthetic steps in grh biosynthesis, the genes grhA, grhB, grhC, grhE, grhT and grhQ were cloned for heterologous expression in the host organism S. albus. S. albus strains expressing the grh minimal PKS with and without the grh cyclases did not result in detectable amounts of polyketides or shunt products. This is in high contrast to other type II PKS systems. In an alternative approach, all cyclase genes were individually deleted withinin the grh cluster. Based on these results, the enzyme GrhE does not seem to play a significant role in the grh biosynthesis since griseorhodin A was still produced. The deletion of the other cyclases, GrhT and GrhQ, abolished the biosynthesis and no polyketides were detected. This suggests that a multienzyme complex is required for early polyketide assembly. To further investigate early griseorhodin A biosynthesis, two other griseorhodin A gene deletion mutants, ΔgrhH-P (deletion of all post-PKS genes) and ΔgrhH-Q (deletion of all post-PKS genes and a cyclase gene), were generated during the present study. The ΔgrhH-P mutant contained only the proposed early griseorhodin genes. Heterologous expression of this construct led to the production of a complex mixture of compounds. It produced identical intermediates to the ΔgrhM mutant, which was generated and expressed during a previous study and for which an early oxygenation role is proposed during griseorhodin A biosynthesis. Four compounds from the ΔgrhH-P mutant expression were isolated and analysed indicating potentially new early intermediates. However, in sufficient material was isolated for structure elucidation by 2D NMR.
To further investigate whether a functional protein complex is relevant at the early stage of griseorhodin A biosynthesis, in the second mutant ΔgrhH-Q, the cyclase GrhQ was additionally deleted, and the expression led to abolishment of griseorhodin A biosynthesis. This was in agreement with the GrhQ inactivation study. These results indicate and further support the presence of a multienzyme complex during the course of griseorhodin A biosynthesis. To date, griseorhodin A is one of the longest aromatic polyketides and therefore, could require the synergistic actions of cyclases (GrhQ) and other stabilising enzymes such as GrhS for correct polyketide assembly.
In order to gain deeper insights into the enzymatic activities responsible for griseorhodin A biosynthesis, two constructs with single gene deletions within the cluster, constructed in a previous study were further investigated. The two deleted genes, grhU and grhV encode proteins with unknown function. In the present study, these modified clusters were subsequently heterologously expressed in S. albus and analysed by high performance liquid chromatography (HPLC), high resolution mass spectroscopy (HRMS) and cryogenic or capillary NMR for the production of secondary metabolites. Heterologous expression of both mutants resulted in complex mixtures of compounds, and 21 pure compounds were isolated by various chromatographic methods. The structure of one of the compounds originating from the ΔgrhV gene deletion mutant was elucidated from 500 μg of sample and the structure revealed the decarboxylated 13,13´- fused tridecaketide dimer. The structure suggests that GrhV is responsible for an oxidative modification in the griseorhodin A molecule. To our knowledge, the present study gives first insights into the oxygenase function of GrhV and its homologues found in other pentangular biosynthetic pathways.},
url = {https://hdl.handle.net/20.500.11811/5283}
}
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