Investigations on structure and biosynthesis of ribosomal peptide natural products involved in a tripartite mutualistic interaction
Investigations on structure and biosynthesis of ribosomal peptide natural products involved in a tripartite mutualistic interaction
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DissertationDate of Exam
12.12.2023Date of Publication
16.04.2024Advisor
König, Gabriele M.Co-Referee
Dahl, ChristianeMetadata
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Abstract
Ribosomally synthesized and post-translationally modified peptides (RiPPs) constitute a chemically diverse group of biologically active molecules. Several RiPPs play outstanding roles in the social behavior and physiology of microorganisms. Their biosynthesis is a complex and energetically costly process that is strictly orchestrated in the bacterial cell. The timing and level of expression of bacterial RiPP biosynthetic gene clusters (BGCs) are typically controlled by multiple regulatory pathways that respond to environmental changes, microbial interactions, stress, or master switches during specific growth phases.
Streptomyces griseus S4-7 was isolated from the rhizosphere of strawberry plants as the representative strain producing uncharacterized metabolites with inhibitory effect against the phytopathogenic fungus Fusarium oxysporum. The S4-7 strain was further found to be engaged in a mutualistic interaction with plants and pollinator bees where bacteria provide protection from pathogens and the honeybees serve as vectors for effective dispersal.
The present thesis provides insights into the structures and biosynthesis of ribosomal peptide natural products implicated in the protective effect of S. griseus S4-7. These metabolites are encoded in two BGCs. The cpr BGC encodes an uncharacterized thiopeptide predicted to be the largest member of this subfamily of RiPPs ever reported. The second BGC, here termed mrsS47, which stands for mixed RiPP system, encodes five precursor peptides varying in length and sequence. Since both cryptic BGCs were found to be silent under standard cultivation conditions in the laboratory, several strategies including elicitation and overexpression of pleiotropic and cluster-situated regulators were applied in the natural producer S4-7. As none of these efforts led to the production of the target metabolites, the cpr and mrsS47 BGCs were independently investigated.
The cpr BGC was cloned and refactored for heterologous expression in Streptomyces. Constitutive and inducible promoters as well as three different Streptomyces species were tested for production of the thiopeptide. An [M+2H]2+ ion at m/z 1070.08 was ultimately obtained as a cpr BGC–related metabolite that still needs to be confirmed as the mature thiopeptide.
On the other hand, the mrsS47 BGC was studied through an in vitro/in vivo reconstitution approach. The five precursor peptides (MrsA1-A5) and the six biosynthetic enzymes (MrsM, MrsKC, MrsP, MrsJ, MrsY, and MrsR) were heterologously expressed in E. coli, purified, and subsequently used for reconstitution of the main biosynthetic steps. It was experimentally shown that the five peptides encoded in the mrsS47 BGC are independently modified and belong to different subclasses of RiPPs. MrsA1 and MrsA2 were confirmed to be modified by the lanthipeptide synthetase MrsM and the oxidoreductase MrsJ giving linear peptides containing dehydroamino acids and, in the case of MrsA2, D-alanine and D-amino butyric acid residues. The class III lanthipeptide synthetase MrsKC installed a labionin structure in MrsA3 and MrsA4. The S8 serine protease MrsP was proven to be responsible for the maturation of three of the five peptides (MrsA3-A5). Finally, MrsA5 was found to be modified by the radical SAM protein and the iron-containing redox enzyme (MrsY and MrsR, respectively) and is proposed to be the effector of a standalone peptide-processing biological conflict system. Further bioinformatics analyses revealed that the mrs BGC of S. griseus S4-7 is a member of a widespread family of mixed RiPP systems from Actinobacteria. These mrs BGCs share a highly conserved operon likely encoding a peptide-processing biological conflict system, and at least one additional modified peptide. The mrsS47 is thus the first member of this newly discovered family of mixed RiPP BGCs to be experimentally characterized.
Streptomyces griseus S4-7 was isolated from the rhizosphere of strawberry plants as the representative strain producing uncharacterized metabolites with inhibitory effect against the phytopathogenic fungus Fusarium oxysporum. The S4-7 strain was further found to be engaged in a mutualistic interaction with plants and pollinator bees where bacteria provide protection from pathogens and the honeybees serve as vectors for effective dispersal.
The present thesis provides insights into the structures and biosynthesis of ribosomal peptide natural products implicated in the protective effect of S. griseus S4-7. These metabolites are encoded in two BGCs. The cpr BGC encodes an uncharacterized thiopeptide predicted to be the largest member of this subfamily of RiPPs ever reported. The second BGC, here termed mrsS47, which stands for mixed RiPP system, encodes five precursor peptides varying in length and sequence. Since both cryptic BGCs were found to be silent under standard cultivation conditions in the laboratory, several strategies including elicitation and overexpression of pleiotropic and cluster-situated regulators were applied in the natural producer S4-7. As none of these efforts led to the production of the target metabolites, the cpr and mrsS47 BGCs were independently investigated.
The cpr BGC was cloned and refactored for heterologous expression in Streptomyces. Constitutive and inducible promoters as well as three different Streptomyces species were tested for production of the thiopeptide. An [M+2H]2+ ion at m/z 1070.08 was ultimately obtained as a cpr BGC–related metabolite that still needs to be confirmed as the mature thiopeptide.
On the other hand, the mrsS47 BGC was studied through an in vitro/in vivo reconstitution approach. The five precursor peptides (MrsA1-A5) and the six biosynthetic enzymes (MrsM, MrsKC, MrsP, MrsJ, MrsY, and MrsR) were heterologously expressed in E. coli, purified, and subsequently used for reconstitution of the main biosynthetic steps. It was experimentally shown that the five peptides encoded in the mrsS47 BGC are independently modified and belong to different subclasses of RiPPs. MrsA1 and MrsA2 were confirmed to be modified by the lanthipeptide synthetase MrsM and the oxidoreductase MrsJ giving linear peptides containing dehydroamino acids and, in the case of MrsA2, D-alanine and D-amino butyric acid residues. The class III lanthipeptide synthetase MrsKC installed a labionin structure in MrsA3 and MrsA4. The S8 serine protease MrsP was proven to be responsible for the maturation of three of the five peptides (MrsA3-A5). Finally, MrsA5 was found to be modified by the radical SAM protein and the iron-containing redox enzyme (MrsY and MrsR, respectively) and is proposed to be the effector of a standalone peptide-processing biological conflict system. Further bioinformatics analyses revealed that the mrs BGC of S. griseus S4-7 is a member of a widespread family of mixed RiPP systems from Actinobacteria. These mrs BGCs share a highly conserved operon likely encoding a peptide-processing biological conflict system, and at least one additional modified peptide. The mrsS47 is thus the first member of this newly discovered family of mixed RiPP BGCs to be experimentally characterized.
Subjects
Bacterial Natural Products, Streptomyces griseus, Ribosomally synthesized and post-translationally modified peptides (RiPPs), thiopeptides, lanthipeptides, mixed RiPP system, cluster-situated regulators (CSRs)
Classification (DDC)
500 Naturwissenschaften 570 Biowissenschaften, BiologieSoto Zarazua, Maria Guadalupe: Investigations on structure and biosynthesis of ribosomal peptide natural products involved in a tripartite mutualistic interaction. - Bonn, 2024. - Dissertation, Rheinische Friedrich-Wilhelms-Universität Bonn.
Online-Ausgabe in bonndoc: https://nbn-resolving.org/urn:nbn:de:hbz:5-75511
Online-Ausgabe in bonndoc: https://nbn-resolving.org/urn:nbn:de:hbz:5-75511
@phdthesis{handle:20.500.11811/11491,
urn: https://nbn-resolving.org/urn:nbn:de:hbz:5-75511,
doi: https://doi.org/10.48565/bonndoc-263,
author = {{Maria Guadalupe Soto Zarazua}},
title = {Investigations on structure and biosynthesis of ribosomal peptide natural products involved in a tripartite mutualistic interaction},
school = {Rheinische Friedrich-Wilhelms-Universität Bonn},
year = 2024,
month = apr,
note = {Ribosomally synthesized and post-translationally modified peptides (RiPPs) constitute a chemically diverse group of biologically active molecules. Several RiPPs play outstanding roles in the social behavior and physiology of microorganisms. Their biosynthesis is a complex and energetically costly process that is strictly orchestrated in the bacterial cell. The timing and level of expression of bacterial RiPP biosynthetic gene clusters (BGCs) are typically controlled by multiple regulatory pathways that respond to environmental changes, microbial interactions, stress, or master switches during specific growth phases.
Streptomyces griseus S4-7 was isolated from the rhizosphere of strawberry plants as the representative strain producing uncharacterized metabolites with inhibitory effect against the phytopathogenic fungus Fusarium oxysporum. The S4-7 strain was further found to be engaged in a mutualistic interaction with plants and pollinator bees where bacteria provide protection from pathogens and the honeybees serve as vectors for effective dispersal.
The present thesis provides insights into the structures and biosynthesis of ribosomal peptide natural products implicated in the protective effect of S. griseus S4-7. These metabolites are encoded in two BGCs. The cpr BGC encodes an uncharacterized thiopeptide predicted to be the largest member of this subfamily of RiPPs ever reported. The second BGC, here termed mrsS47, which stands for mixed RiPP system, encodes five precursor peptides varying in length and sequence. Since both cryptic BGCs were found to be silent under standard cultivation conditions in the laboratory, several strategies including elicitation and overexpression of pleiotropic and cluster-situated regulators were applied in the natural producer S4-7. As none of these efforts led to the production of the target metabolites, the cpr and mrsS47 BGCs were independently investigated.
The cpr BGC was cloned and refactored for heterologous expression in Streptomyces. Constitutive and inducible promoters as well as three different Streptomyces species were tested for production of the thiopeptide. An [M+2H]2+ ion at m/z 1070.08 was ultimately obtained as a cpr BGC–related metabolite that still needs to be confirmed as the mature thiopeptide.
On the other hand, the mrsS47 BGC was studied through an in vitro/in vivo reconstitution approach. The five precursor peptides (MrsA1-A5) and the six biosynthetic enzymes (MrsM, MrsKC, MrsP, MrsJ, MrsY, and MrsR) were heterologously expressed in E. coli, purified, and subsequently used for reconstitution of the main biosynthetic steps. It was experimentally shown that the five peptides encoded in the mrsS47 BGC are independently modified and belong to different subclasses of RiPPs. MrsA1 and MrsA2 were confirmed to be modified by the lanthipeptide synthetase MrsM and the oxidoreductase MrsJ giving linear peptides containing dehydroamino acids and, in the case of MrsA2, D-alanine and D-amino butyric acid residues. The class III lanthipeptide synthetase MrsKC installed a labionin structure in MrsA3 and MrsA4. The S8 serine protease MrsP was proven to be responsible for the maturation of three of the five peptides (MrsA3-A5). Finally, MrsA5 was found to be modified by the radical SAM protein and the iron-containing redox enzyme (MrsY and MrsR, respectively) and is proposed to be the effector of a standalone peptide-processing biological conflict system. Further bioinformatics analyses revealed that the mrs BGC of S. griseus S4-7 is a member of a widespread family of mixed RiPP systems from Actinobacteria. These mrs BGCs share a highly conserved operon likely encoding a peptide-processing biological conflict system, and at least one additional modified peptide. The mrsS47 is thus the first member of this newly discovered family of mixed RiPP BGCs to be experimentally characterized.},
url = {https://hdl.handle.net/20.500.11811/11491}
}
urn: https://nbn-resolving.org/urn:nbn:de:hbz:5-75511,
doi: https://doi.org/10.48565/bonndoc-263,
author = {{Maria Guadalupe Soto Zarazua}},
title = {Investigations on structure and biosynthesis of ribosomal peptide natural products involved in a tripartite mutualistic interaction},
school = {Rheinische Friedrich-Wilhelms-Universität Bonn},
year = 2024,
month = apr,
note = {Ribosomally synthesized and post-translationally modified peptides (RiPPs) constitute a chemically diverse group of biologically active molecules. Several RiPPs play outstanding roles in the social behavior and physiology of microorganisms. Their biosynthesis is a complex and energetically costly process that is strictly orchestrated in the bacterial cell. The timing and level of expression of bacterial RiPP biosynthetic gene clusters (BGCs) are typically controlled by multiple regulatory pathways that respond to environmental changes, microbial interactions, stress, or master switches during specific growth phases.
Streptomyces griseus S4-7 was isolated from the rhizosphere of strawberry plants as the representative strain producing uncharacterized metabolites with inhibitory effect against the phytopathogenic fungus Fusarium oxysporum. The S4-7 strain was further found to be engaged in a mutualistic interaction with plants and pollinator bees where bacteria provide protection from pathogens and the honeybees serve as vectors for effective dispersal.
The present thesis provides insights into the structures and biosynthesis of ribosomal peptide natural products implicated in the protective effect of S. griseus S4-7. These metabolites are encoded in two BGCs. The cpr BGC encodes an uncharacterized thiopeptide predicted to be the largest member of this subfamily of RiPPs ever reported. The second BGC, here termed mrsS47, which stands for mixed RiPP system, encodes five precursor peptides varying in length and sequence. Since both cryptic BGCs were found to be silent under standard cultivation conditions in the laboratory, several strategies including elicitation and overexpression of pleiotropic and cluster-situated regulators were applied in the natural producer S4-7. As none of these efforts led to the production of the target metabolites, the cpr and mrsS47 BGCs were independently investigated.
The cpr BGC was cloned and refactored for heterologous expression in Streptomyces. Constitutive and inducible promoters as well as three different Streptomyces species were tested for production of the thiopeptide. An [M+2H]2+ ion at m/z 1070.08 was ultimately obtained as a cpr BGC–related metabolite that still needs to be confirmed as the mature thiopeptide.
On the other hand, the mrsS47 BGC was studied through an in vitro/in vivo reconstitution approach. The five precursor peptides (MrsA1-A5) and the six biosynthetic enzymes (MrsM, MrsKC, MrsP, MrsJ, MrsY, and MrsR) were heterologously expressed in E. coli, purified, and subsequently used for reconstitution of the main biosynthetic steps. It was experimentally shown that the five peptides encoded in the mrsS47 BGC are independently modified and belong to different subclasses of RiPPs. MrsA1 and MrsA2 were confirmed to be modified by the lanthipeptide synthetase MrsM and the oxidoreductase MrsJ giving linear peptides containing dehydroamino acids and, in the case of MrsA2, D-alanine and D-amino butyric acid residues. The class III lanthipeptide synthetase MrsKC installed a labionin structure in MrsA3 and MrsA4. The S8 serine protease MrsP was proven to be responsible for the maturation of three of the five peptides (MrsA3-A5). Finally, MrsA5 was found to be modified by the radical SAM protein and the iron-containing redox enzyme (MrsY and MrsR, respectively) and is proposed to be the effector of a standalone peptide-processing biological conflict system. Further bioinformatics analyses revealed that the mrs BGC of S. griseus S4-7 is a member of a widespread family of mixed RiPP systems from Actinobacteria. These mrs BGCs share a highly conserved operon likely encoding a peptide-processing biological conflict system, and at least one additional modified peptide. The mrsS47 is thus the first member of this newly discovered family of mixed RiPP BGCs to be experimentally characterized.},
url = {https://hdl.handle.net/20.500.11811/11491}
}
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