Expression of Terpene Synthases from Edible Fungi using Yeast Chassis
(2024) KMBM05 20241Applied Microbiology
- Abstract
- Secondary metabolites produced from organisms are compounds that are not directly connected to the growth of organisms but that serve many important purposes for them in nature. Terpenes and terpenoids form a section of the secondary metabolites, produced from terpene synthase (TPS) enzymes. Fungal species are highly dependent on secondary metabolites and especially the terpenoid compounds for numerous adaptive tasks, such as defense and formation of symbiotic relationships. Despite the importance of terpenes and terpenoids in fungi and the large number of fungal species compared to plant species, the corresponding TPS genes in fungal genomes are much less studied than those from plants. In this work, as part of a larger study at UCPH,... (More)
- Secondary metabolites produced from organisms are compounds that are not directly connected to the growth of organisms but that serve many important purposes for them in nature. Terpenes and terpenoids form a section of the secondary metabolites, produced from terpene synthase (TPS) enzymes. Fungal species are highly dependent on secondary metabolites and especially the terpenoid compounds for numerous adaptive tasks, such as defense and formation of symbiotic relationships. Despite the importance of terpenes and terpenoids in fungi and the large number of fungal species compared to plant species, the corresponding TPS genes in fungal genomes are much less studied than those from plants. In this work, as part of a larger study at UCPH, unexplored TPSs from edible fungal species were studied, to contribute to the enzyme characterizations and product exploration. 31 TPSs enzymes from fungal genomes of shiitake mushroom Lentinula edodes, oyster mushroom Pleurotus ostreatus, porcini mushroom Boletus edulis, jelly fungus Auricularia subglabra and cheese fungi Penicillium roqueforti, Penicillium biforme, and Penicillium camemberti were expressed. The genes were introduced in yeast through multicopy plasmids, using the uracil-specific excision reagent (USER) cloning technique to construct the plasmids together with an inducible GAL1 promoter. The products were analyzed in vivo with headspace solid phase microextraction (HS-SPME) as well as in vitro with hexane extraction, using gas chromatography mass spectrometry (GC-MS). From the results it could be concluded that three TPSs produced mainly monoterpenes, nine TPSs mainly sesquiterpenes, and one TPS mainly diterpenes. One putative sesquiterpene without a provided name was detected as well as sesquiterpenes sinularene and myltayl-4(12)-ene which could not be found in fungal species. The monoterpene synthase (mono-TPSs) belonged in majority to the Ascomycota phylum and the sesquiterpene synthase (sesqui-TPSs) in majority to the Basidiomycota phylum. The catalytic activity of the TPS genes was traced back to the phylogenetic tree, where especially TPSs producing monoterpenes located in one cluster and TPSs producing sesquiterpenes of cadalane structures in another. An additional experiment with expression of Erg20p(N127W), a gene described to accumulate GPP in yeast cells, led to an unexpected increase in sesquiterpenes. Furthermore, a third of the transformants were induced into buffered medium (pH 6.5) to analyze the correlation between pH and enzymatic activity. The buffered induction led to an overall greater sesquiterpene production for all except the three tested inactive transformants that still showed no terpene peaks. (Less)
- Popular Abstract
- Fungal species exist everywhere on earth, highly important in various types of ecosystems. There are many sorts of fungi, including the toxic molds growing on bread, the yeast used for beer production, and mushrooms that can be picked in the forest. Fungal species can be utilized for many purposes, where a large aspect is their role in the food sector. They are included in dishes or used in food industries, such as for various meat substitutes and cheese production. Fungi produce a great array of small molecules, providing the species with specific bioactivities. One group of the small molecules are the terpene hydrocarbons and their modified structures termed terpenoids. The compounds have been less studied in fungi compared to plants,... (More)
- Fungal species exist everywhere on earth, highly important in various types of ecosystems. There are many sorts of fungi, including the toxic molds growing on bread, the yeast used for beer production, and mushrooms that can be picked in the forest. Fungal species can be utilized for many purposes, where a large aspect is their role in the food sector. They are included in dishes or used in food industries, such as for various meat substitutes and cheese production. Fungi produce a great array of small molecules, providing the species with specific bioactivities. One group of the small molecules are the terpene hydrocarbons and their modified structures termed terpenoids. The compounds have been less studied in fungi compared to plants, and the understanding of product formations is low. However, as terpenes and terpenoids can carry a variety of properties, such as antimicrobial, anti-inflammatory, and anticancer, and fungal species are highly consumed by humans, the interactions between the produced terpenes and terpenoids with the human microbiota is important.
This thesis project was part of a larger study at University of Copenhagen, aiming to provide knowledge on the terpene production from edible fungi. Yeast cells were used to express the terpene-producing enzymes TPSs. GC-MS was used to analyze the formed products. TPSs from 4 edible mushrooms, specifically shiitake mushroom, oyster mushroom, porcini mushroom, and jelly fungi, as well as 3 cheese fungi were studied. Not all TPSs demonstrated activity, but the active presented either one or a few terpene products, or they were able to produce a greater range of terpenes. A large portion of the detected terpenes had been discovered in fungi before, some in edible species and some in the same species, but sesquiterpenes not described in fungi were also produced. The work studied how pH affected the activity of the TPSs hence the terpene production. This thesis work provided insights into terpenes formed from edible fungi, allowing continuous studies on potential terpenoid productions. (Less)
Please use this url to cite or link to this publication:
http://lup.lub.lu.se/student-papers/record/9165227
- author
- Buhrgard, Helena LU
- supervisor
- organization
- alternative title
- Uttryck av terpensyntaser från ätbar svamp i jästceller
- course
- KMBM05 20241
- year
- 2024
- type
- H2 - Master's Degree (Two Years)
- subject
- keywords
- Terpenes, Terpene synthases, Terpenoids, Edible fungi, Yeast chassis, GC-MS, USER cloning, Applied microbiology
- language
- English
- id
- 9165227
- date added to LUP
- 2024-06-20 14:15:38
- date last changed
- 2024-06-20 14:15:38
@misc{9165227,
abstract = {{Secondary metabolites produced from organisms are compounds that are not directly connected to the growth of organisms but that serve many important purposes for them in nature. Terpenes and terpenoids form a section of the secondary metabolites, produced from terpene synthase (TPS) enzymes. Fungal species are highly dependent on secondary metabolites and especially the terpenoid compounds for numerous adaptive tasks, such as defense and formation of symbiotic relationships. Despite the importance of terpenes and terpenoids in fungi and the large number of fungal species compared to plant species, the corresponding TPS genes in fungal genomes are much less studied than those from plants. In this work, as part of a larger study at UCPH, unexplored TPSs from edible fungal species were studied, to contribute to the enzyme characterizations and product exploration. 31 TPSs enzymes from fungal genomes of shiitake mushroom Lentinula edodes, oyster mushroom Pleurotus ostreatus, porcini mushroom Boletus edulis, jelly fungus Auricularia subglabra and cheese fungi Penicillium roqueforti, Penicillium biforme, and Penicillium camemberti were expressed. The genes were introduced in yeast through multicopy plasmids, using the uracil-specific excision reagent (USER) cloning technique to construct the plasmids together with an inducible GAL1 promoter. The products were analyzed in vivo with headspace solid phase microextraction (HS-SPME) as well as in vitro with hexane extraction, using gas chromatography mass spectrometry (GC-MS). From the results it could be concluded that three TPSs produced mainly monoterpenes, nine TPSs mainly sesquiterpenes, and one TPS mainly diterpenes. One putative sesquiterpene without a provided name was detected as well as sesquiterpenes sinularene and myltayl-4(12)-ene which could not be found in fungal species. The monoterpene synthase (mono-TPSs) belonged in majority to the Ascomycota phylum and the sesquiterpene synthase (sesqui-TPSs) in majority to the Basidiomycota phylum. The catalytic activity of the TPS genes was traced back to the phylogenetic tree, where especially TPSs producing monoterpenes located in one cluster and TPSs producing sesquiterpenes of cadalane structures in another. An additional experiment with expression of Erg20p(N127W), a gene described to accumulate GPP in yeast cells, led to an unexpected increase in sesquiterpenes. Furthermore, a third of the transformants were induced into buffered medium (pH 6.5) to analyze the correlation between pH and enzymatic activity. The buffered induction led to an overall greater sesquiterpene production for all except the three tested inactive transformants that still showed no terpene peaks.}},
author = {{Buhrgard, Helena}},
language = {{eng}},
note = {{Student Paper}},
title = {{Expression of Terpene Synthases from Edible Fungi using Yeast Chassis}},
year = {{2024}},
}