The santalene synthase from Cinnamomum camphora: Reconstruction of a sesquiterpene synthase from a monoterpene synthase

Girolamo, Alice Di; Durairaj, Janani; Houwelingen, Adèle van; Verstappen, Francel; Bosch, Dirk; Cankar, Katarina; Bouwmeester, Harro J.; Ridder, Dick de; Dijk, Aalt D. J. van; Beekwilder, Jules

doi:10.1016/j.abb.2020.108647

Cited by 12 publications

(13 citation statements)

References 52 publications

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“…Besides, SauSSy from S . austrocaledonicum 13 , SspiSSy from S. spicatum 13 and CiCaSSy from Cinnamomum camphora 16 were characterized to possess the product distributions similar to that of SaSSy, and SanSyn from Clausena lansium was found to be a product-specific enzyme, with α -santalene as its predominant product (Fig. 1 ) 17 .…”

Section: Introductionmentioning

confidence: 97%

Rationally engineering santalene synthase to readjust the component ratio of sandalwood oil

et al. 2022

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Plant essential oils (PEOs) are widely used in cosmetic and nutraceutical industries. The component ratios of PEOs determine their qualities. Controlling the component ratios is challenging in construction of PEO biotechnological platforms. Here, we explore the catalytic reaction pathways of both product-promiscuous and product-specific santalene synthases (i.e., SaSSy and SanSyn) by multiscale simulations. F441 of SanSyn is found as a key residue restricting the conformational dynamics of the intermediates, and thereby the direct deprotonation by the general base T298 dominantly produce α-santalene. The subsequent mutagenesis of this plastic residue leads to generation of a mutant enzyme SanSynF441V which can produce both α- and β-santalenes. Through metabolic engineering efforts, the santalene/santalol titer reaches 704.2 mg/L and the component ratio well matches the ISO 3518:2002 standard. This study represents a paradigm of constructing biotechnological platforms of PEOs with desirable component ratios by the combination of metabolic and enzymatic engineering.

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Section: Introductionmentioning

confidence: 97%

Rationally engineering santalene synthase to readjust the component ratio of sandalwood oil

et al. 2022

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“…An Escherichia coli strain derived from BL21(DE3), harbouring a plasmid expressing all genes necessary for the synthesis of farnesyl diphosphate (FDP) (pBbE5k‐MevT (CO)‐MBIS (CO)), 24 was used for the production of valencene. C. nootkatensis valencene synthase (CnVS) 25 was chosen as a target for this experiment, together with a small library of single mutants (plasmids were kindly provided by Isobionics BV) (Table 1).…”

Section: Methodsmentioning

confidence: 99%

“…C. nootkatensis valencene synthase (CnVS) 25 was chosen as a target for this experiment, together with a small library of single mutants (plasmids were kindly provided by Isobionics BV) (Table 1). Synthases were expressed using the inducible plasmid pACYCDuet‐1, as previously described 24 …”

Section: Methodsmentioning

confidence: 99%

“…Fermentations were performed as previously described. 24 In short, pre-cultures were grown in 5 mL LB medium supplemented with 50 μg/mL kanamycin and 50 μg/mL chloramphenicol in 50 mL Greiner tubes. Subsequently, the pre-cultures were diluted to an OD600 of 0.150 in 20 mL 2xYT medium with antibiotics in 100 mL glass flasks and incubated at 37 C 250 rpm until an OD600 of 0.4-0.5 was reached.…”

Section: Cultivation Conditions For Gc-ms Analysismentioning

confidence: 99%

“…GC-MS analysis was performed as described before. 24 In brief, a 7980A GC system combined with a 5975C inert MSD detector (70 eV) (Agilent Technologies, Netherlands) was used. The system is equipped with a 7683 auto-sampler and injector and a Phenomenex Zebron ZB-5-ms column of 30 m length Â 0.25 mm internal diameter and 0.25 μm stationary phase, with a Guardian precolumn (5 m).…”

Section: Gc-ms Measurements and Data Analysismentioning

confidence: 99%

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The use of proton transfer reaction mass spectrometry for high throughput screening of terpene synthases

Di Girolamo,

Pedrotti,

Koot

et al. 2023

J Mass Spectrom

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In this work, we introduce the application of proton transfer reaction mass spectrometry (PTR‐MS) for the selection of improved terpene synthase mutants. In comparison with gas chromatography mass spectrometry (GC‐MS)‐based methods, PTR‐MS could offer advantages by reduction of sample preparation steps and analysis time. The method we propose here allows for minimal sample preparation and analysis time and provides a promising platform for the high throughput screening (HTS) of large enzyme mutant libraries. To investigate the feasibility of a PTR‐MS‐based screening method, we employed a small library of Callitropsis nootkatensis valencene synthase (CnVS) mutants. Bacterial cultures expressing enzyme mutants were subjected to different growth formats, and headspace terpenes concentrations measured by PTR‐Qi‐ToF‐MS were compared with GC‐MS, to rank the activity of the enzyme mutants. For all cultivation formats, including 96 deep well plates, PTR‐Qi‐ToF‐MS resulted in the same ranking of the enzyme variants, compared with the canonical format using 100 mL flasks and GC‐MS analysis. This study provides a first basis for the application of rapid PTR‐Qi‐ToF‐MS detection, in combination with multi‐well formats, in HTS screening methods for the selection of highly productive terpene synthases.

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Sandalwood Fragrance Pathway and Its Engineering for Sustainable Production of High-Value Santalols

Narayanan

Nagegowda

2022

Compendium of Plant Genomes

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The santalene synthase from Cinnamomum camphora: Reconstruction of a sesquiterpene synthase from a monoterpene synthase

Cited by 12 publications

References 52 publications

Rationally engineering santalene synthase to readjust the component ratio of sandalwood oil

Rationally engineering santalene synthase to readjust the component ratio of sandalwood oil

The use of proton transfer reaction mass spectrometry for high throughput screening of terpene synthases

Sandalwood Fragrance Pathway and Its Engineering for Sustainable Production of High-Value Santalols

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