Use of pantothenate as a metabolic switch increases the genetic stability of farnesene producing Saccharomyces cerevisiae

Sandoval, Celeste M.; Ayson, Marites; Moss, Nathan A.; Lieu, Bonny; Jackson, Peter; Gaucher, Sara P.; Horning, Tizita; Dahl, Robert H.; Denery, Judith R.; Abbott, Derek A.; Meadows, Adam L.

doi:10.1016/j.ymben.2014.07.006

Cited by 57 publications

(42 citation statements)

References 35 publications

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“…Cessation of product formation and growth in prolonged fermentations at low cell concentrations and below the maximal triterpenoid load per cell observed under nitrogen‐limitation indicates decreased cell viability due to accumulation of toxic intermediates or reactive oxygen species as has been reported for production of farnesene and artemisinic acid (Paddon et al, ; Sandoval et al, ). Formation of reactive oxygen species through uncoupling of NADPH oxidation and substrate oxidation is commonly observed in monooxygenase‐type reactions and can severely affect cell viability by damaging proteins and destabilizing membranes (Blank, Ebert, Buehler, & Bühler, ; Farrugia & Balzan, ; Perrone, Tan, & Dawes, ; Zangar, Davydov, & Verma, ).…”

Section: Discussionmentioning

confidence: 81%

Fermentation and purification strategies for the production of betulinic acid and its lupane‐type precursors in Saccharomyces cerevisiae

Czarnotta

Dianat

Korf

et al. 2017

Biotech & Bioengineering

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Microbial production of plant derived, biologically active compounds has the potential to provide economic and ecologic alternatives to existing low productive, plant-based processes. Current production of the pharmacologically active cyclic triterpenoid betulinic acid is realized by extraction from the bark of plane tree or birch. Here, we reengineered the reported betulinic acid pathway into Saccharomyces cerevisiae and used this novel strain to develop efficient fermentation and product purification methods. Fed-batch cultivations with ethanol excess, using either an ethanol-pulse feed or controlling a constant ethanol concentration in the fermentation medium, significantly enhanced production of betulinic acid and its triterpenoid precursors. The beneficial effect of excess ethanol was further exploited in nitrogen-limited resting cell fermentations, yielding betulinic acid concentrations of 182 mg/L, and total triterpenoid concentrations of 854 mg/L, the highest concentrations reported so far. Purification of lupane-type triterpenoids with high selectivity and yield was achieved by solid-liquid extraction without prior cell disruption using polar aprotic solvents such as acetone or ethyl acetate and subsequent precipitation with strong acids. This study highlights the potential of microbial production of plant derived triterpenoids in S. cerevisiae by combining metabolic and process engineering.

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Section: Discussionmentioning

confidence: 81%

Fermentation and purification strategies for the production of betulinic acid and its lupane‐type precursors in Saccharomyces cerevisiae

Czarnotta

Dianat

Korf

et al. 2017

Biotech & Bioengineering

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“…One possibility is that pantothenate acts as primary source for the MF biosynthesis pathway. In the budding yeast Saccharomyces cerevisiae , pantothenate is known to be the limiting precursor of CoA synthesis 24 . Furthermore, a recent study revealed that the concentration of pantothenate in the culture medium can dramatically affect the production rate of farnesene, a sesquiterpene isoprenoid polymer of acetyl-CoA, which is synthesized via the mevalonate pathway 25 .…”

Section: Discussionmentioning

confidence: 99%

Metabolomics reveals an involvement of pantothenate for male production responding to the short-day stimulus in the water flea, Daphnia pulex

Toyota

Gavin

Miyagawa

et al. 2016

Sci Rep

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Under favorable conditions, the micro-crustacean Daphnia pulex produces female offspring by parthenogenesis, whereas under unfavorable conditions, they produce male offspring to induce sexual reproduction (environmental sex determination: ESD). We recently established a suitable system for ESD studies using D. pulex WTN6 strain, in which the sex of the offspring can be regulated by alterations in day-length; long-day and short-day conditions can induce female and male offspring, respectively. Taking advantage of this system, we have already demonstrated that methyl farnesoate (MF) synthesis is necessary for male offspring production, and identified ionotropic glutamate receptors as an upstream regulator of MF signaling. Despite these findings, the molecular mechanisms associated with MF signaling have not yet been well elucidated. In this study, we analyzed the whole metabolic profiles of mother daphnids reared under long-day (female-producing) and short-day (male-producing) conditions, and discovered that pantothenate (vitamin B5), a known precursor to coenzyme A, was significantly accumulated in response to the short-day condition. To confirm the innate role of pantothenate in D. pulex, this metabolite was administered to mother daphnids resulting in a significantly increased proportion of male offspring producing mothers. This study provides novel insights of the metabolic mechanisms of the ESD system in D. pulex.

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“…Production of sesquiterpenes could be significantly enhanced by improving acetyl-CoA synthesis in combination with overexpression of the endogenous ERG10 [2]. On the other hand, acetoacetyl-CoA thiolase from Clostridium acetobutylicum , which has been previously used for production of farnesene [27], was shown to have a significantly higher in vitro activity compared to the thiolase from S. cerevisiae [29]. Besides, further genetic modifications might be required to pull more flux through the mevalonate pathway and particularly to redirect flux from the FPP branch point towards farnesene.…”

Section: Discussionmentioning

confidence: 99%

Effects of acetoacetyl-CoA synthase expression on production of farnesene in Saccharomyces cerevisiae

Tippmann

Ferreira

Siewers

et al. 2017

Journal of Industrial Microbiology and Biotechnology

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Efficient production of sesquiterpenes in Saccharomyces cerevisiae requires a high flux through the mevalonate pathway. To achieve this, the supply of acetyl-CoA plays a crucial role, partially because nine moles of acetyl-CoA are necessary to produce one mole of farnesyl diphosphate, but also to overcome the thermodynamic constraint imposed on the first reaction, in which acetoacetyl-CoA is produced from two moles of acetyl-CoA by acetoacetyl-CoA thiolase. Recently, a novel acetoacetyl-CoA synthase (nphT7) has been identified from Streptomyces sp. strain CL190, which catalyzes the irreversible condensation of malonyl-CoA and acetyl-CoA to acetoacetyl-CoA and, therefore, represents a potential target to increase the flux through the mevalonate pathway. This study investigates the effect of acetoacetyl-CoA synthase on growth as well as the production of farnesene and compares different homologs regarding their efficiency. While plasmid-based expression of nphT7 did not improve final farnesene titers, the construction of an alternative pathway, which exclusively relies on the malonyl-CoA bypass, was detrimental for growth and farnesene production. The presented results indicate that the overall functionality of the bypass was limited by the efficiency of acetoacetyl-CoA synthase (nphT7). Besides modulation of the expression level, which could be used as a means to partially restore the phenotype, nphT7 from Streptomyces glaucescens showed clearly higher efficiency compared to Streptomyces sp. strain CL190.Electronic supplementary materialThe online version of this article (doi:10.1007/s10295-017-1911-6) contains supplementary material, which is available to authorized users.

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Use of pantothenate as a metabolic switch increases the genetic stability of farnesene producing Saccharomyces cerevisiae

Cited by 57 publications

References 35 publications

Fermentation and purification strategies for the production of betulinic acid and its lupane‐type precursors in Saccharomyces cerevisiae

Fermentation and purification strategies for the production of betulinic acid and its lupane‐type precursors in Saccharomyces cerevisiae

Metabolomics reveals an involvement of pantothenate for male production responding to the short-day stimulus in the water flea, Daphnia pulex

Effects of acetoacetyl-CoA synthase expression on production of farnesene in Saccharomyces cerevisiae

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