Esterification of geraniol as a strategy for increasing product titre and specificity in engineered Escherichia coli

Micaela G Chacón, Alice Marriott, Emanuele G Kendrick, Matthew Q Styles, David J Leak

Research output: Contribution to journalArticle

Abstract

BACKGROUND: Geraniol, an acyclic monoterpene alcohol, is found as a primary constituent in the essential oils of plants such as geranium, lemongrass and rose. The floral-like scent of geraniol has made it a popular constituent of flavour and fragrance products. Over recent decades biotechnology has made significant progress towards the development of industrial platforms for the production of commercially valuable monoterpenoids, such as geraniol, through expression of recombinant terpene biosynthetic pathways in microbial hosts. Titres, however, have been hindered due to the inherent toxicity of these compounds-which are often utilised for anti-microbial and anti-fungal functions in their host plant.

RESULTS: In this study we modified an Escherichia coli strain, engineered to express a heterologous mevalonate pathway, by replacement of the terpene synthase with a geraniol synthase from Ocimum basilicum for the production of geraniol, and co-expressed an alcohol acyltransferase (AAT) from Rosa hybrida for the specific acetylation of geraniol. The low water solubility of geranyl acetate facilitated its partition into the organic phase of a two-phase system, relieving the cellular toxicity attributed to the build-up of geraniol in the aqueous phase. In a partially optimised system this strain produced 4.8 g/L geranyl acetate (based on the aqueous volume) which, on a molar equivalent basis, represents the highest monoterpene titre achieved from microbial culture to date. It was also found that esterification of geraniol prevented bioconversion into other monoterpenoids, leading to a significant improvement in product specificity, with geranyl acetate being the sole product observed.

CONCLUSION: In this study we have shown that it is possible to both overcome the toxicity limit impeding the production of the monoterpene alcohol geraniol and mitigate product loss in culture through endogenous metabolism by using an in vivo esterification strategy. This strategy has resulted in the highest geraniol (equivalent) titres achieved from a microbial host, and presents esterification as a viable approach to increasing the titres obtained in microbial monoterpenoid production.

LanguageEnglish
Article number105
Number of pages11
JournalMicrobial Cell Factories
Volume18
Issue number1
DOIs
StatusPublished - 8 Jun 2019

Keywords

  • Geranyl acetate
  • Geraniol
  • Monoterpene

Cite this

Esterification of geraniol as a strategy for increasing product titre and specificity in engineered Escherichia coli. / Chacón, Micaela G; Marriott, Alice; Kendrick, Emanuele G; Styles, Matthew Q; Leak, David J.

In: Microbial Cell Factories, Vol. 18, No. 1, 105, 08.06.2019.

Research output: Contribution to journalArticle

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AU - Chacón, Micaela G

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AU - Kendrick, Emanuele G

AU - Styles, Matthew Q

AU - Leak, David J

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N2 - BACKGROUND: Geraniol, an acyclic monoterpene alcohol, is found as a primary constituent in the essential oils of plants such as geranium, lemongrass and rose. The floral-like scent of geraniol has made it a popular constituent of flavour and fragrance products. Over recent decades biotechnology has made significant progress towards the development of industrial platforms for the production of commercially valuable monoterpenoids, such as geraniol, through expression of recombinant terpene biosynthetic pathways in microbial hosts. Titres, however, have been hindered due to the inherent toxicity of these compounds-which are often utilised for anti-microbial and anti-fungal functions in their host plant.RESULTS: In this study we modified an Escherichia coli strain, engineered to express a heterologous mevalonate pathway, by replacement of the terpene synthase with a geraniol synthase from Ocimum basilicum for the production of geraniol, and co-expressed an alcohol acyltransferase (AAT) from Rosa hybrida for the specific acetylation of geraniol. The low water solubility of geranyl acetate facilitated its partition into the organic phase of a two-phase system, relieving the cellular toxicity attributed to the build-up of geraniol in the aqueous phase. In a partially optimised system this strain produced 4.8 g/L geranyl acetate (based on the aqueous volume) which, on a molar equivalent basis, represents the highest monoterpene titre achieved from microbial culture to date. It was also found that esterification of geraniol prevented bioconversion into other monoterpenoids, leading to a significant improvement in product specificity, with geranyl acetate being the sole product observed.CONCLUSION: In this study we have shown that it is possible to both overcome the toxicity limit impeding the production of the monoterpene alcohol geraniol and mitigate product loss in culture through endogenous metabolism by using an in vivo esterification strategy. This strategy has resulted in the highest geraniol (equivalent) titres achieved from a microbial host, and presents esterification as a viable approach to increasing the titres obtained in microbial monoterpenoid production.

AB - BACKGROUND: Geraniol, an acyclic monoterpene alcohol, is found as a primary constituent in the essential oils of plants such as geranium, lemongrass and rose. The floral-like scent of geraniol has made it a popular constituent of flavour and fragrance products. Over recent decades biotechnology has made significant progress towards the development of industrial platforms for the production of commercially valuable monoterpenoids, such as geraniol, through expression of recombinant terpene biosynthetic pathways in microbial hosts. Titres, however, have been hindered due to the inherent toxicity of these compounds-which are often utilised for anti-microbial and anti-fungal functions in their host plant.RESULTS: In this study we modified an Escherichia coli strain, engineered to express a heterologous mevalonate pathway, by replacement of the terpene synthase with a geraniol synthase from Ocimum basilicum for the production of geraniol, and co-expressed an alcohol acyltransferase (AAT) from Rosa hybrida for the specific acetylation of geraniol. The low water solubility of geranyl acetate facilitated its partition into the organic phase of a two-phase system, relieving the cellular toxicity attributed to the build-up of geraniol in the aqueous phase. In a partially optimised system this strain produced 4.8 g/L geranyl acetate (based on the aqueous volume) which, on a molar equivalent basis, represents the highest monoterpene titre achieved from microbial culture to date. It was also found that esterification of geraniol prevented bioconversion into other monoterpenoids, leading to a significant improvement in product specificity, with geranyl acetate being the sole product observed.CONCLUSION: In this study we have shown that it is possible to both overcome the toxicity limit impeding the production of the monoterpene alcohol geraniol and mitigate product loss in culture through endogenous metabolism by using an in vivo esterification strategy. This strategy has resulted in the highest geraniol (equivalent) titres achieved from a microbial host, and presents esterification as a viable approach to increasing the titres obtained in microbial monoterpenoid production.

KW - Geranyl acetate

KW - Geraniol

KW - Monoterpene

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VL - 18

JO - Microbial Cell Factories

T2 - Microbial Cell Factories

JF - Microbial Cell Factories

SN - 1475-2859

IS - 1

M1 - 105

ER -