Metabolic engineering of Ashbya gossypii for deciphering the de novo biosynthesis of γ-lactones

Detalhes bibliográficos
Autor(a) principal: Silva, Rui
Data de Publicação: 2019
Outros Autores: Aguiar, Tatiana Quinta, Coelho, Eduardo, Jiménez, Alberto, Revuelta, José Luis, Domingues, Lucília
Tipo de documento: Artigo
Idioma: eng
Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
Texto Completo: http://hdl.handle.net/1822/60401
Resumo: Background Lactones are highly valuable cyclic esters of hydroxy fatty acids that find application as pure fragrances or as building blocks of speciality chemicals. While chemical synthesis often leads to undesired racemic mixtures, microbial production allows obtaining optically pure lactones. The production of a specific lactone by biotransformation depends on the supply of the corresponding hydroxy fatty acid, which has economic and industrial value similar to γ-lactones. Hence, the identification and exploration of microorganisms with the rare natural ability for de novo biosynthesis of lactones will contribute to the long-term sustainability of microbial production. In this study, the innate ability of Ashbya gossypii for de novo production of γ-lactones from glucose was evaluated and improved. Results Characterization of the volatile organic compounds produced by nine strains of this industrial filamentous fungus in glucose-based medium revealed the noteworthy presence of seven chemically different γ-lactones. To decipher and understand the de novo biosynthesis of γ-lactones from glucose, we developed metabolic engineering strategies focused on the fatty acid biosynthesis and the β-oxidation pathways. Overexpression of AgDES589, encoding a desaturase for the conversion of oleic acid (C18:1) into linoleic acid (C18:2), and deletion of AgELO624, which encodes an elongase that catalyses the formation of C20:0 and C22:0 fatty acids, greatly increased the production of γ-lactones (up to 6.4-fold; (7.6 ± 0.8) × 103 µg/gCell Dry Weight). Further substitution of AgPOX1, encoding the exclusive acyl-CoA oxidase in A. gossypii, by a codon-optimized POX2 gene from Yarrowia lipolytica, which encodes a specific long chain acyl-CoA oxidase, fine-tuned the biosynthesis of γ-decalactone to a relative production of more than 99%. Conclusions This study demonstrates the potential of A. gossypii as a model and future platform for de novo biosynthesis of γ-lactones. By means of metabolic engineering, key enzymatic steps involved in their production were elucidated. Moreover, the combinatorial metabolic engineering strategies developed resulted in improved de novo biosynthesis of γ-decalactone. In sum, these proof-of-concept data revealed yet unknown metabolic and genetic determinants important for the future exploration of the de novo production of γ-lactones as an alternative to biotransformation processes.
id RCAP_ada8ff48d8c6e7bbfe522e89b3d7f2af
oai_identifier_str oai:repositorium.sdum.uminho.pt:1822/60401
network_acronym_str RCAP
network_name_str Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
repository_id_str 7160
spelling Metabolic engineering of Ashbya gossypii for deciphering the de novo biosynthesis of γ-lactonesVolatile organic compoundsγ-LactonesAshbya gossypiDe novo biosynthesisMetabolic engineeringFatty acid biosynthesisß-Oxidationgamma-LactonesAshbya gossypiiβ-OxidationScience & TechnologyBackground Lactones are highly valuable cyclic esters of hydroxy fatty acids that find application as pure fragrances or as building blocks of speciality chemicals. While chemical synthesis often leads to undesired racemic mixtures, microbial production allows obtaining optically pure lactones. The production of a specific lactone by biotransformation depends on the supply of the corresponding hydroxy fatty acid, which has economic and industrial value similar to γ-lactones. Hence, the identification and exploration of microorganisms with the rare natural ability for de novo biosynthesis of lactones will contribute to the long-term sustainability of microbial production. In this study, the innate ability of Ashbya gossypii for de novo production of γ-lactones from glucose was evaluated and improved. Results Characterization of the volatile organic compounds produced by nine strains of this industrial filamentous fungus in glucose-based medium revealed the noteworthy presence of seven chemically different γ-lactones. To decipher and understand the de novo biosynthesis of γ-lactones from glucose, we developed metabolic engineering strategies focused on the fatty acid biosynthesis and the β-oxidation pathways. Overexpression of AgDES589, encoding a desaturase for the conversion of oleic acid (C18:1) into linoleic acid (C18:2), and deletion of AgELO624, which encodes an elongase that catalyses the formation of C20:0 and C22:0 fatty acids, greatly increased the production of γ-lactones (up to 6.4-fold; (7.6 ± 0.8) × 103 µg/gCell Dry Weight). Further substitution of AgPOX1, encoding the exclusive acyl-CoA oxidase in A. gossypii, by a codon-optimized POX2 gene from Yarrowia lipolytica, which encodes a specific long chain acyl-CoA oxidase, fine-tuned the biosynthesis of γ-decalactone to a relative production of more than 99%. Conclusions This study demonstrates the potential of A. gossypii as a model and future platform for de novo biosynthesis of γ-lactones. By means of metabolic engineering, key enzymatic steps involved in their production were elucidated. Moreover, the combinatorial metabolic engineering strategies developed resulted in improved de novo biosynthesis of γ-decalactone. In sum, these proof-of-concept data revealed yet unknown metabolic and genetic determinants important for the future exploration of the de novo production of γ-lactones as an alternative to biotransformation processes.This study was supported by: the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2019 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684) (Post-Doc grant to T. Q. Aguiar and Ph.D. grant to E. Coelho), Ph.D. Grant PD/BD/113812/2015 to R. Silva (Doctoral Program in Applied and Environmental Microbiology), and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020-Programa Operacional Regional do Norte; the Spanish Ministerio de Economía y Competitividad (BIO2014-56930-P and BIO2017-88435-R) and Junta de Castilla y León (SA016P17) to A. Jiménez and J.L. Revuelta.info:eu-repo/semantics/publishedVersionSpringer NatureUniversidade do MinhoSilva, RuiAguiar, Tatiana QuintaCoelho, EduardoJiménez, AlbertoRevuelta, José LuisDomingues, Lucília2019-122019-12-01T00:00:00Zinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articleapplication/pdfhttp://hdl.handle.net/1822/60401engSilva, Rui; Aguiar, Tatiana Q.; Coelho, Eduardo; Jiménez, Alberto; Revuelta, José Luis; Domingues, Lucília, Metabolic engineering of Ashbya gossypii for deciphering the de novo biosynthesis of -lactones. Microbial Cell Factories, 18(62), 20191475-28591475-285910.1186/s12934-019-1113-130922300http://www.microbialcellfactories.com/info:eu-repo/semantics/openAccessreponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãoinstacron:RCAAP2023-07-21T12:19:44Zoai:repositorium.sdum.uminho.pt:1822/60401Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-19T19:12:42.379578Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãofalse
dc.title.none.fl_str_mv Metabolic engineering of Ashbya gossypii for deciphering the de novo biosynthesis of γ-lactones
title Metabolic engineering of Ashbya gossypii for deciphering the de novo biosynthesis of γ-lactones
spellingShingle Metabolic engineering of Ashbya gossypii for deciphering the de novo biosynthesis of γ-lactones
Silva, Rui
Volatile organic compounds
γ-Lactones
Ashbya gossypi
De novo biosynthesis
Metabolic engineering
Fatty acid biosynthesis
ß-Oxidation
gamma-Lactones
Ashbya gossypii
β-Oxidation
Science & Technology
title_short Metabolic engineering of Ashbya gossypii for deciphering the de novo biosynthesis of γ-lactones
title_full Metabolic engineering of Ashbya gossypii for deciphering the de novo biosynthesis of γ-lactones
title_fullStr Metabolic engineering of Ashbya gossypii for deciphering the de novo biosynthesis of γ-lactones
title_full_unstemmed Metabolic engineering of Ashbya gossypii for deciphering the de novo biosynthesis of γ-lactones
title_sort Metabolic engineering of Ashbya gossypii for deciphering the de novo biosynthesis of γ-lactones
author Silva, Rui
author_facet Silva, Rui
Aguiar, Tatiana Quinta
Coelho, Eduardo
Jiménez, Alberto
Revuelta, José Luis
Domingues, Lucília
author_role author
author2 Aguiar, Tatiana Quinta
Coelho, Eduardo
Jiménez, Alberto
Revuelta, José Luis
Domingues, Lucília
author2_role author
author
author
author
author
dc.contributor.none.fl_str_mv Universidade do Minho
dc.contributor.author.fl_str_mv Silva, Rui
Aguiar, Tatiana Quinta
Coelho, Eduardo
Jiménez, Alberto
Revuelta, José Luis
Domingues, Lucília
dc.subject.por.fl_str_mv Volatile organic compounds
γ-Lactones
Ashbya gossypi
De novo biosynthesis
Metabolic engineering
Fatty acid biosynthesis
ß-Oxidation
gamma-Lactones
Ashbya gossypii
β-Oxidation
Science & Technology
topic Volatile organic compounds
γ-Lactones
Ashbya gossypi
De novo biosynthesis
Metabolic engineering
Fatty acid biosynthesis
ß-Oxidation
gamma-Lactones
Ashbya gossypii
β-Oxidation
Science & Technology
description Background Lactones are highly valuable cyclic esters of hydroxy fatty acids that find application as pure fragrances or as building blocks of speciality chemicals. While chemical synthesis often leads to undesired racemic mixtures, microbial production allows obtaining optically pure lactones. The production of a specific lactone by biotransformation depends on the supply of the corresponding hydroxy fatty acid, which has economic and industrial value similar to γ-lactones. Hence, the identification and exploration of microorganisms with the rare natural ability for de novo biosynthesis of lactones will contribute to the long-term sustainability of microbial production. In this study, the innate ability of Ashbya gossypii for de novo production of γ-lactones from glucose was evaluated and improved. Results Characterization of the volatile organic compounds produced by nine strains of this industrial filamentous fungus in glucose-based medium revealed the noteworthy presence of seven chemically different γ-lactones. To decipher and understand the de novo biosynthesis of γ-lactones from glucose, we developed metabolic engineering strategies focused on the fatty acid biosynthesis and the β-oxidation pathways. Overexpression of AgDES589, encoding a desaturase for the conversion of oleic acid (C18:1) into linoleic acid (C18:2), and deletion of AgELO624, which encodes an elongase that catalyses the formation of C20:0 and C22:0 fatty acids, greatly increased the production of γ-lactones (up to 6.4-fold; (7.6 ± 0.8) × 103 µg/gCell Dry Weight). Further substitution of AgPOX1, encoding the exclusive acyl-CoA oxidase in A. gossypii, by a codon-optimized POX2 gene from Yarrowia lipolytica, which encodes a specific long chain acyl-CoA oxidase, fine-tuned the biosynthesis of γ-decalactone to a relative production of more than 99%. Conclusions This study demonstrates the potential of A. gossypii as a model and future platform for de novo biosynthesis of γ-lactones. By means of metabolic engineering, key enzymatic steps involved in their production were elucidated. Moreover, the combinatorial metabolic engineering strategies developed resulted in improved de novo biosynthesis of γ-decalactone. In sum, these proof-of-concept data revealed yet unknown metabolic and genetic determinants important for the future exploration of the de novo production of γ-lactones as an alternative to biotransformation processes.
publishDate 2019
dc.date.none.fl_str_mv 2019-12
2019-12-01T00:00:00Z
dc.type.status.fl_str_mv info:eu-repo/semantics/publishedVersion
dc.type.driver.fl_str_mv info:eu-repo/semantics/article
format article
status_str publishedVersion
dc.identifier.uri.fl_str_mv http://hdl.handle.net/1822/60401
url http://hdl.handle.net/1822/60401
dc.language.iso.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv Silva, Rui; Aguiar, Tatiana Q.; Coelho, Eduardo; Jiménez, Alberto; Revuelta, José Luis; Domingues, Lucília, Metabolic engineering of Ashbya gossypii for deciphering the de novo biosynthesis of -lactones. Microbial Cell Factories, 18(62), 2019
1475-2859
1475-2859
10.1186/s12934-019-1113-1
30922300
http://www.microbialcellfactories.com/
dc.rights.driver.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv application/pdf
dc.publisher.none.fl_str_mv Springer Nature
publisher.none.fl_str_mv Springer Nature
dc.source.none.fl_str_mv reponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação
instacron:RCAAP
instname_str Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação
instacron_str RCAAP
institution RCAAP
reponame_str Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
collection Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
repository.name.fl_str_mv Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação
repository.mail.fl_str_mv
_version_ 1799132563376177152