Cargando…

Dynamic Control of ERG20 and ERG9 Expression for Improved Casbene Production in Saccharomyces cerevisiae

Production of plant metabolites in microbial hosts represents a promising alternative to traditional chemical-based methods. Diterpenoids are compounds with interesting applications as pharmaceuticals, fragrances and biomaterials. Casbene, in particular, serves as a precursor to many complex diterpe...

Descripción completa

Detalles Bibliográficos
Autores principales: Callari, Roberta, Meier, Yvan, Ravasio, Davide, Heider, Harald
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6221901/
https://www.ncbi.nlm.nih.gov/pubmed/30443546
http://dx.doi.org/10.3389/fbioe.2018.00160
_version_ 1783369104681336832
author Callari, Roberta
Meier, Yvan
Ravasio, Davide
Heider, Harald
author_facet Callari, Roberta
Meier, Yvan
Ravasio, Davide
Heider, Harald
author_sort Callari, Roberta
collection PubMed
description Production of plant metabolites in microbial hosts represents a promising alternative to traditional chemical-based methods. Diterpenoids are compounds with interesting applications as pharmaceuticals, fragrances and biomaterials. Casbene, in particular, serves as a precursor to many complex diterpenoids found in plants from the Euphorbiaceae family that have shown potential therapeutic effects. Here, we engineered the budding yeast Saccharomyces cerevisiae for improved biosynthesis of the diterpene casbene. We first expressed, in yeast, a geranylgeranyl diphosphate synthase from Phomopsys amygdali in order to boost the geranylgeranyl diphosphate pool inside the cells. The enzyme uses isopentenyl diphosphate and dimethylallyl diphosphate to directly generate geranylgeranyl diphosphate. When co-expressing a casbene synthase from Ricinus communis the yeast was able to produce casbene in the order of 30 mg/L. Redirecting the flux from FPP and sterols, by means of the ergosterol sensitive promoter of ERG1, allowed for plasmid-based casbene production of 81.4 mg/L. Integration of the target genes into the yeast genome, together with the replacement of the promoter regions of ERG20 and ERG9 with combinations of ergosterol- and glucose-sensitive promoters, generated a titer of 108.5 mg/L of casbene. We here succeeded to engineer an improved route for geranylgeranyl diphosphate synthesis in yeast. Furthermore, we showed that the concurrent dynamic control of ERG20 and ERG9 expression, using ergosterol and carbon source regulation mechanisms, could substantially improve diterpene titer. Our approach will pave the way for a more sustainable production of GGPP- and casbene-derived products.
format Online
Article
Text
id pubmed-6221901
institution National Center for Biotechnology Information
language English
publishDate 2018
publisher Frontiers Media S.A.
record_format MEDLINE/PubMed
spelling pubmed-62219012018-11-15 Dynamic Control of ERG20 and ERG9 Expression for Improved Casbene Production in Saccharomyces cerevisiae Callari, Roberta Meier, Yvan Ravasio, Davide Heider, Harald Front Bioeng Biotechnol Bioengineering and Biotechnology Production of plant metabolites in microbial hosts represents a promising alternative to traditional chemical-based methods. Diterpenoids are compounds with interesting applications as pharmaceuticals, fragrances and biomaterials. Casbene, in particular, serves as a precursor to many complex diterpenoids found in plants from the Euphorbiaceae family that have shown potential therapeutic effects. Here, we engineered the budding yeast Saccharomyces cerevisiae for improved biosynthesis of the diterpene casbene. We first expressed, in yeast, a geranylgeranyl diphosphate synthase from Phomopsys amygdali in order to boost the geranylgeranyl diphosphate pool inside the cells. The enzyme uses isopentenyl diphosphate and dimethylallyl diphosphate to directly generate geranylgeranyl diphosphate. When co-expressing a casbene synthase from Ricinus communis the yeast was able to produce casbene in the order of 30 mg/L. Redirecting the flux from FPP and sterols, by means of the ergosterol sensitive promoter of ERG1, allowed for plasmid-based casbene production of 81.4 mg/L. Integration of the target genes into the yeast genome, together with the replacement of the promoter regions of ERG20 and ERG9 with combinations of ergosterol- and glucose-sensitive promoters, generated a titer of 108.5 mg/L of casbene. We here succeeded to engineer an improved route for geranylgeranyl diphosphate synthesis in yeast. Furthermore, we showed that the concurrent dynamic control of ERG20 and ERG9 expression, using ergosterol and carbon source regulation mechanisms, could substantially improve diterpene titer. Our approach will pave the way for a more sustainable production of GGPP- and casbene-derived products. Frontiers Media S.A. 2018-11-01 /pmc/articles/PMC6221901/ /pubmed/30443546 http://dx.doi.org/10.3389/fbioe.2018.00160 Text en Copyright © 2018 Callari, Meier, Ravasio and Heider. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Bioengineering and Biotechnology
Callari, Roberta
Meier, Yvan
Ravasio, Davide
Heider, Harald
Dynamic Control of ERG20 and ERG9 Expression for Improved Casbene Production in Saccharomyces cerevisiae
title Dynamic Control of ERG20 and ERG9 Expression for Improved Casbene Production in Saccharomyces cerevisiae
title_full Dynamic Control of ERG20 and ERG9 Expression for Improved Casbene Production in Saccharomyces cerevisiae
title_fullStr Dynamic Control of ERG20 and ERG9 Expression for Improved Casbene Production in Saccharomyces cerevisiae
title_full_unstemmed Dynamic Control of ERG20 and ERG9 Expression for Improved Casbene Production in Saccharomyces cerevisiae
title_short Dynamic Control of ERG20 and ERG9 Expression for Improved Casbene Production in Saccharomyces cerevisiae
title_sort dynamic control of erg20 and erg9 expression for improved casbene production in saccharomyces cerevisiae
topic Bioengineering and Biotechnology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6221901/
https://www.ncbi.nlm.nih.gov/pubmed/30443546
http://dx.doi.org/10.3389/fbioe.2018.00160
work_keys_str_mv AT callariroberta dynamiccontroloferg20anderg9expressionforimprovedcasbeneproductioninsaccharomycescerevisiae
AT meieryvan dynamiccontroloferg20anderg9expressionforimprovedcasbeneproductioninsaccharomycescerevisiae
AT ravasiodavide dynamiccontroloferg20anderg9expressionforimprovedcasbeneproductioninsaccharomycescerevisiae
AT heiderharald dynamiccontroloferg20anderg9expressionforimprovedcasbeneproductioninsaccharomycescerevisiae