Production of terpenes in Aspergillus nidulans: Engineering of the secondary metabolite gene cluster: Dissertation

Kirsi Bromann

Research output: ThesisDissertation

Abstract

Secondary metabolites, or natural products, are produced by a variety of microorganisms and plants but are particularly abundant in filamentous fungi. Secondary metabolites can be toxic or have pharmaceutical applications as antibiotics, anticancer, antiparasitic and antifungal agents. Terpenes are a large group of secondary metabolites with wide array of bioactivities. Filamentous fungus, Aspergillus nidulans, is a fungal model organism and a close relative to other industrially relevant Aspergillus species. A. nidulans is a known producer of many bioactive metabolites, including polyketides, nonribosomal peptides and sesquiterpenes. Sequencing of A. nidulans genome has revealed numerous secondary metabolite gene clusters, yet products of many of these biosynthetic pathways are unknown since the expression of the clustered genes usually remains silent in laboratory conditions. Finding ways to induce the gene expression of silent biosynthetic clusters can lead to discovery of novel metabolites with unknown bioactivities. Furthermore, identifying key biosynthetic genes in interesting metabolite pathways is a prerequisite for genetic engineering of heterologous production systems. In this study, we wanted to explore the terpene producing ability of A. nidulans. We discovered a novel diterpene, ent-pimara-8(14),15-diene (PD), in A. nidulans by predicting a biosynthetic gene cluster with genomic mining. We identified a Zn(II)2Cys6-type transcription factor, PbcR, and demonstrated its role as a positive regulator for the predicted gene cluster. The transcriptional profile of the pbcR overexpression strain (oe:PbcR) was analyzed by Real-Time Quantitative Reverse Transcription PCR (qRT-PCR) and DNA microarray. Overexpression of pbcR led to upregulation of the predicted biosynthetic cluster genes and subsequent PD production in A. nidulans. In addition to PD cluster genes, transcriptional changes were observed for a number of secondary metabolite genes in oe:PbcR. Microscopy of the plate cultures revealed changes in the oe:PbcR morphology. To study the bioactivity of PD, we developed and optimized a PD extraction method for the engineered A. nidulans production strain. An accelerated solvent extraction (ASE) with a combined High-Performance-Liquid-Chromatography (HPLC) purification enabled us to collect highly pure PD for the Nuclear Magnetic Resonance (NMR) analysis and bioactivity testing. While PD did not exhibit inhibitory function against Staphylococcus aureus or Leishmania donovani, we observed an antioxidant activity for PD in a radical scavenging assay. We also characterized A. nidulans PD synthase (PbcA) with overexpression and deletion assays and showed that it is indispensable for PD production in A. nidulans. Furthermore, we extended our genetic engineering to study the potential of A. nidulans as a heterologous host for mono- and diterpene production. Overexpression of Fusarium fujikuroi ent-kaurene synthase (cps/ks) and Citrus unshiu gamma-terpinene synthase resulted in ent-kaurene and gamma-terpinene production, respectively. The results reported in this thesis highlight the advantage of using genomic mining in the search for novel biosynthetic pathways. Additionally, this thesis presents a proof-of-concept study for using A. nidulans as heterologous terpene production host.
Original languageEnglish
QualificationDoctor Degree
Awarding Institution
  • University of Helsinki
Supervisors/Advisors
  • Nakari-Setälä, Tiina, Supervisor
  • Ruohonen, Laura, Supervisor
Award date6 May 2016
Place of PublicationEspoo
Publisher
Print ISBNs978-951-38-8414-7
Electronic ISBNs978-951-38-8415-4
Publication statusPublished - 2016
MoE publication typeG5 Doctoral dissertation (article)

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Aspergillus nidulans
Terpenes
Multigene Family
Genetic Engineering
Diterpenes
Biosynthetic Pathways
Fungi
Kaurane Diterpenes
Polyketides
Gene Expression
Antiparasitic Agents
Leishmania donovani
Monoterpenes
Sesquiterpenes
Citrus
Antifungal Agents
Poisons
Fusarium
Aspergillus
Regulator Genes

Cite this

Bromann, Kirsi. / Production of terpenes in Aspergillus nidulans : Engineering of the secondary metabolite gene cluster: Dissertation. Espoo : VTT Technical Research Centre of Finland, 2016. 118 p.
@phdthesis{d853854957744029abe466798fa9f83f,
title = "Production of terpenes in Aspergillus nidulans: Engineering of the secondary metabolite gene cluster: Dissertation",
abstract = "Secondary metabolites, or natural products, are produced by a variety of microorganisms and plants but are particularly abundant in filamentous fungi. Secondary metabolites can be toxic or have pharmaceutical applications as antibiotics, anticancer, antiparasitic and antifungal agents. Terpenes are a large group of secondary metabolites with wide array of bioactivities. Filamentous fungus, Aspergillus nidulans, is a fungal model organism and a close relative to other industrially relevant Aspergillus species. A. nidulans is a known producer of many bioactive metabolites, including polyketides, nonribosomal peptides and sesquiterpenes. Sequencing of A. nidulans genome has revealed numerous secondary metabolite gene clusters, yet products of many of these biosynthetic pathways are unknown since the expression of the clustered genes usually remains silent in laboratory conditions. Finding ways to induce the gene expression of silent biosynthetic clusters can lead to discovery of novel metabolites with unknown bioactivities. Furthermore, identifying key biosynthetic genes in interesting metabolite pathways is a prerequisite for genetic engineering of heterologous production systems. In this study, we wanted to explore the terpene producing ability of A. nidulans. We discovered a novel diterpene, ent-pimara-8(14),15-diene (PD), in A. nidulans by predicting a biosynthetic gene cluster with genomic mining. We identified a Zn(II)2Cys6-type transcription factor, PbcR, and demonstrated its role as a positive regulator for the predicted gene cluster. The transcriptional profile of the pbcR overexpression strain (oe:PbcR) was analyzed by Real-Time Quantitative Reverse Transcription PCR (qRT-PCR) and DNA microarray. Overexpression of pbcR led to upregulation of the predicted biosynthetic cluster genes and subsequent PD production in A. nidulans. In addition to PD cluster genes, transcriptional changes were observed for a number of secondary metabolite genes in oe:PbcR. Microscopy of the plate cultures revealed changes in the oe:PbcR morphology. To study the bioactivity of PD, we developed and optimized a PD extraction method for the engineered A. nidulans production strain. An accelerated solvent extraction (ASE) with a combined High-Performance-Liquid-Chromatography (HPLC) purification enabled us to collect highly pure PD for the Nuclear Magnetic Resonance (NMR) analysis and bioactivity testing. While PD did not exhibit inhibitory function against Staphylococcus aureus or Leishmania donovani, we observed an antioxidant activity for PD in a radical scavenging assay. We also characterized A. nidulans PD synthase (PbcA) with overexpression and deletion assays and showed that it is indispensable for PD production in A. nidulans. Furthermore, we extended our genetic engineering to study the potential of A. nidulans as a heterologous host for mono- and diterpene production. Overexpression of Fusarium fujikuroi ent-kaurene synthase (cps/ks) and Citrus unshiu gamma-terpinene synthase resulted in ent-kaurene and gamma-terpinene production, respectively. The results reported in this thesis highlight the advantage of using genomic mining in the search for novel biosynthetic pathways. Additionally, this thesis presents a proof-of-concept study for using A. nidulans as heterologous terpene production host.",
author = "Kirsi Bromann",
note = "BA3111 54 p. + app. 64 p.",
year = "2016",
language = "English",
isbn = "978-951-38-8414-7",
series = "VTT Science",
publisher = "VTT Technical Research Centre of Finland",
number = "126",
address = "Finland",
school = "University of Helsinki",

}

Production of terpenes in Aspergillus nidulans : Engineering of the secondary metabolite gene cluster: Dissertation. / Bromann, Kirsi.

Espoo : VTT Technical Research Centre of Finland, 2016. 118 p.

Research output: ThesisDissertation

TY - THES

T1 - Production of terpenes in Aspergillus nidulans

T2 - Engineering of the secondary metabolite gene cluster: Dissertation

AU - Bromann, Kirsi

N1 - BA3111 54 p. + app. 64 p.

PY - 2016

Y1 - 2016

N2 - Secondary metabolites, or natural products, are produced by a variety of microorganisms and plants but are particularly abundant in filamentous fungi. Secondary metabolites can be toxic or have pharmaceutical applications as antibiotics, anticancer, antiparasitic and antifungal agents. Terpenes are a large group of secondary metabolites with wide array of bioactivities. Filamentous fungus, Aspergillus nidulans, is a fungal model organism and a close relative to other industrially relevant Aspergillus species. A. nidulans is a known producer of many bioactive metabolites, including polyketides, nonribosomal peptides and sesquiterpenes. Sequencing of A. nidulans genome has revealed numerous secondary metabolite gene clusters, yet products of many of these biosynthetic pathways are unknown since the expression of the clustered genes usually remains silent in laboratory conditions. Finding ways to induce the gene expression of silent biosynthetic clusters can lead to discovery of novel metabolites with unknown bioactivities. Furthermore, identifying key biosynthetic genes in interesting metabolite pathways is a prerequisite for genetic engineering of heterologous production systems. In this study, we wanted to explore the terpene producing ability of A. nidulans. We discovered a novel diterpene, ent-pimara-8(14),15-diene (PD), in A. nidulans by predicting a biosynthetic gene cluster with genomic mining. We identified a Zn(II)2Cys6-type transcription factor, PbcR, and demonstrated its role as a positive regulator for the predicted gene cluster. The transcriptional profile of the pbcR overexpression strain (oe:PbcR) was analyzed by Real-Time Quantitative Reverse Transcription PCR (qRT-PCR) and DNA microarray. Overexpression of pbcR led to upregulation of the predicted biosynthetic cluster genes and subsequent PD production in A. nidulans. In addition to PD cluster genes, transcriptional changes were observed for a number of secondary metabolite genes in oe:PbcR. Microscopy of the plate cultures revealed changes in the oe:PbcR morphology. To study the bioactivity of PD, we developed and optimized a PD extraction method for the engineered A. nidulans production strain. An accelerated solvent extraction (ASE) with a combined High-Performance-Liquid-Chromatography (HPLC) purification enabled us to collect highly pure PD for the Nuclear Magnetic Resonance (NMR) analysis and bioactivity testing. While PD did not exhibit inhibitory function against Staphylococcus aureus or Leishmania donovani, we observed an antioxidant activity for PD in a radical scavenging assay. We also characterized A. nidulans PD synthase (PbcA) with overexpression and deletion assays and showed that it is indispensable for PD production in A. nidulans. Furthermore, we extended our genetic engineering to study the potential of A. nidulans as a heterologous host for mono- and diterpene production. Overexpression of Fusarium fujikuroi ent-kaurene synthase (cps/ks) and Citrus unshiu gamma-terpinene synthase resulted in ent-kaurene and gamma-terpinene production, respectively. The results reported in this thesis highlight the advantage of using genomic mining in the search for novel biosynthetic pathways. Additionally, this thesis presents a proof-of-concept study for using A. nidulans as heterologous terpene production host.

AB - Secondary metabolites, or natural products, are produced by a variety of microorganisms and plants but are particularly abundant in filamentous fungi. Secondary metabolites can be toxic or have pharmaceutical applications as antibiotics, anticancer, antiparasitic and antifungal agents. Terpenes are a large group of secondary metabolites with wide array of bioactivities. Filamentous fungus, Aspergillus nidulans, is a fungal model organism and a close relative to other industrially relevant Aspergillus species. A. nidulans is a known producer of many bioactive metabolites, including polyketides, nonribosomal peptides and sesquiterpenes. Sequencing of A. nidulans genome has revealed numerous secondary metabolite gene clusters, yet products of many of these biosynthetic pathways are unknown since the expression of the clustered genes usually remains silent in laboratory conditions. Finding ways to induce the gene expression of silent biosynthetic clusters can lead to discovery of novel metabolites with unknown bioactivities. Furthermore, identifying key biosynthetic genes in interesting metabolite pathways is a prerequisite for genetic engineering of heterologous production systems. In this study, we wanted to explore the terpene producing ability of A. nidulans. We discovered a novel diterpene, ent-pimara-8(14),15-diene (PD), in A. nidulans by predicting a biosynthetic gene cluster with genomic mining. We identified a Zn(II)2Cys6-type transcription factor, PbcR, and demonstrated its role as a positive regulator for the predicted gene cluster. The transcriptional profile of the pbcR overexpression strain (oe:PbcR) was analyzed by Real-Time Quantitative Reverse Transcription PCR (qRT-PCR) and DNA microarray. Overexpression of pbcR led to upregulation of the predicted biosynthetic cluster genes and subsequent PD production in A. nidulans. In addition to PD cluster genes, transcriptional changes were observed for a number of secondary metabolite genes in oe:PbcR. Microscopy of the plate cultures revealed changes in the oe:PbcR morphology. To study the bioactivity of PD, we developed and optimized a PD extraction method for the engineered A. nidulans production strain. An accelerated solvent extraction (ASE) with a combined High-Performance-Liquid-Chromatography (HPLC) purification enabled us to collect highly pure PD for the Nuclear Magnetic Resonance (NMR) analysis and bioactivity testing. While PD did not exhibit inhibitory function against Staphylococcus aureus or Leishmania donovani, we observed an antioxidant activity for PD in a radical scavenging assay. We also characterized A. nidulans PD synthase (PbcA) with overexpression and deletion assays and showed that it is indispensable for PD production in A. nidulans. Furthermore, we extended our genetic engineering to study the potential of A. nidulans as a heterologous host for mono- and diterpene production. Overexpression of Fusarium fujikuroi ent-kaurene synthase (cps/ks) and Citrus unshiu gamma-terpinene synthase resulted in ent-kaurene and gamma-terpinene production, respectively. The results reported in this thesis highlight the advantage of using genomic mining in the search for novel biosynthetic pathways. Additionally, this thesis presents a proof-of-concept study for using A. nidulans as heterologous terpene production host.

M3 - Dissertation

SN - 978-951-38-8414-7

T3 - VTT Science

PB - VTT Technical Research Centre of Finland

CY - Espoo

ER -