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3-deoxyachilleol A
(+)-ambrein
8alpha-hydroxypolypoda-13,17,21-triene
onoceranoxide + ((1R,2R,4aS,8aS)-2,5,5,8a-tetramethyl-1-{2-[(1S,4aS,8aS)-5,5,8a-trimethyl-2-methylidenedecahydronaphthalen-1-yl]ethyl}decahydronaphthalen-2-ol
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the enzyme constructs the 6/6/6/6-tetracyclic scaffold baciterpenol A from the head-to-tail monocyclic tetraprenyl-betacurcumene as sesquarterpene cyclase, EC 4.2.1.137, and it also forms the 6/6/7/6/6-pentacyclic onoceranoxide and the 6/6 + 6/6-tetracyclic (1R,2R,4aS,8aS)-2,5,5,8a-tetramethyl-1-[2-[(1S,4aS,8aS)-5,5,8a-trimethyl-2-methylidenedecahydronaphthalen-1-yl]ethyl]decahydron from the tail-to-tail acyclic triterpene squalene via the 6/6-bicyclic intermediate 8alpha-hydroxypolypoda-13,17,21-triene
ratio 64:36
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?
8alpha-hydroxypolypoda-13,17,21-triene
onoceranoxide + 14beta-hydroxyonocera-8(26)-ene
squalene
(+)-ambrein
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via 3-deoxyachilleol A, by mutant BmTC D373C
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?
squalene
3-deoxyarabidiol + H2O
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reaction of all Y167 mutants, no activity with the wild-type enzyme, catalytic mechanism, overview. 3-Deoxyarabidiol, i.e. (5E)-6,10-dimethyl-2-[(3R,3aR,5aS,9aS,9bR)-3a,6,6,9a-tetramethyldodecahydro-1H-cyclopenta[a]naphthalen-3-yl]undeca-5,9-dien-2-ol, is directly synthesized from squalene in the first reaction
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?
squalene
8alpha-hydroxypolypoda-13,17,21-triene
squalene + H2O
8alpha-hydroxypolypoda-13,17,21-triene
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intermediate product
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?
tetraprenyl-beta-curcumene + H2O
baciterpenol A
additional information
?
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3-deoxyachilleol A
(+)-ambrein
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3-deoxyachilleol A
(+)-ambrein
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from squalene
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?
3-deoxyachilleol A
(+)-ambrein
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substrate is produced by AaSHC mutant D377C
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?
8alpha-hydroxypolypoda-13,17,21-triene
onoceranoxide + 14beta-hydroxyonocera-8(26)-ene
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?
8alpha-hydroxypolypoda-13,17,21-triene
onoceranoxide + 14beta-hydroxyonocera-8(26)-ene
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the BmeTC-catalyzed reaction of the substrate is initiated by the protonation of a terminal isopropylidene moiety, triggering a series of cyclization events, erminated by the nucleophilic addition of a hydroxyl group or the deprotonation of H-26 at the resulting C-8 carbocation, respectively
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squalene
8alpha-hydroxypolypoda-13,17,21-triene
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?
squalene
8alpha-hydroxypolypoda-13,17,21-triene
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catalytic mechanism, overview
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?
tetraprenyl-beta-curcumene + H2O
baciterpenol A
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tetraprenyl-beta-curcumene + H2O
baciterpenol A
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catalytic mechanism, overview
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?
additional information
?
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tetraprenyl-beta-curcumene cyclase from Bacillus megaterium is a bifunctional triterpene/sesquarterpene cyclase that converts head-to-tail tetraprenyl-beta-curcumene and tail-to-tail squalene into pentacyclic and bicyclic products, respectively, in vivo. BmeTC has an unprecedented catalytic function in cyclizing squalene from both termini and is an onoceroid synthase. The function of BmeTC enables the synthesis of (+)-ambrein
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additional information
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symmetric and asymmetric onoceroids can be biosynthesized by a single enzyme via an intermediate cyclized at one terminus of squalene
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additional information
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for analysis, the GC-MS method used cannot be used to separate the highly similar compounds such as squalene and 3-deoxyachilleol, or 8alpha-hydroxypolypoda-13,17,21-triene and (+)-ambrein, product analysis by NMR spectrometry
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?
additional information
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onoceroids are synthesized from squalene via an intermediate cyclized from one terminus, such as 8alpha-hydroxypolypoda-13,17,21-triene, by cyclization at both termini. Enzyme BmeTC may have single active site: it starts with the initial cascade leading to the bicyclic intermediate 8alpha-hydroxypolypoda-13,17,21-triene from squalene, then turns molecule 8alpha-hydroxypolypoda-13,17,21-triene outside of the active site cavity, and again uptake 8alpha-hydroxypolypoda-13,17,21-triene to catalyze the formation of additional bicyclic and tricyclic structures of onoceranoxide and 14beta-hydroxyonocera-8(26)-ene on the other side of 8alpha-hydroxypolypoda-13,17,21-triene. BmeTC is not only a bifunctional terpene cyclase which converts tetraprenyl-beta-curcumene and squalene into baciterpenol A and 8alpha-hydroxypolypoda-13,17,21-triene but also an onoceroid synthase that catalyzes the convertion of 8alpha-hydroxypolypoda-13,17,21-triene into onoceranoxide and 14beta-hydroxyonocera-8(26)-ene. GC-MS product analysis, overview
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?
additional information
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the enzyme is a bifunctional triterpene/sesquarterpene cyclase, catalytic reaction mechanism, overview. The wild-type enzyme forms 85.8% 8alpha-hydroxypolypoda-13,17,21-triene, 9.5% onoceranoxide, and 4.7% 14beta-hydroxyonocera-8(26)-ene from squalene, the ratios of mutants are altered. Compounds are identified by NMR spectroscopy
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3-deoxyachilleol A
(+)-ambrein
8alpha-hydroxypolypoda-13,17,21-triene
onoceranoxide + ((1R,2R,4aS,8aS)-2,5,5,8a-tetramethyl-1-{2-[(1S,4aS,8aS)-5,5,8a-trimethyl-2-methylidenedecahydronaphthalen-1-yl]ethyl}decahydronaphthalen-2-ol
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the enzyme constructs the 6/6/6/6-tetracyclic scaffold baciterpenol A from the head-to-tail monocyclic tetraprenyl-betacurcumene as sesquarterpene cyclase, EC 4.2.1.137, and it also forms the 6/6/7/6/6-pentacyclic onoceranoxide and the 6/6 + 6/6-tetracyclic (1R,2R,4aS,8aS)-2,5,5,8a-tetramethyl-1-[2-[(1S,4aS,8aS)-5,5,8a-trimethyl-2-methylidenedecahydronaphthalen-1-yl]ethyl]decahydron from the tail-to-tail acyclic triterpene squalene via the 6/6-bicyclic intermediate 8alpha-hydroxypolypoda-13,17,21-triene
ratio 64:36
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?
8alpha-hydroxypolypoda-13,17,21-triene
onoceranoxide + 14beta-hydroxyonocera-8(26)-ene
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squalene
8alpha-hydroxypolypoda-13,17,21-triene
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squalene + H2O
8alpha-hydroxypolypoda-13,17,21-triene
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intermediate product
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?
tetraprenyl-beta-curcumene + H2O
baciterpenol A
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?
additional information
?
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tetraprenyl-beta-curcumene cyclase from Bacillus megaterium is a bifunctional triterpene/sesquarterpene cyclase that converts head-to-tail tetraprenyl-beta-curcumene and tail-to-tail squalene into pentacyclic and bicyclic products, respectively, in vivo. BmeTC has an unprecedented catalytic function in cyclizing squalene from both termini and is an onoceroid synthase. The function of BmeTC enables the synthesis of (+)-ambrein
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3-deoxyachilleol A
(+)-ambrein
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3-deoxyachilleol A
(+)-ambrein
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from squalene
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?
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D373C
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site-directed mutagenesis, the mutant enzyme BmeTC (D373C) leads to an improved enzyme that is able to catalyze the conversion starting from squalene to 3-deoxyachilleol and further to (+)-ambrein far more efficiently than the described two-enzyme cascade
Y167A
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site-directed mutagenesis, the enzyme mutant produces unnatural tricyclic triterpenol, deoxyarabidiol ((5E)-6,10-dimethyl-2-[(3R,3aR,5aS,9aS,9bR)-3a,6,6,9a-tetramethyldodecahydro-1H-cyclopenta[a]naphthalen-3-yl]undeca-5,9-dien-2-ol), product ratios compared to wild-type, overview
Y167A/L596F
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site-directed mutagenesis, the mutant catalyzes only the synthesis of 8alpha-hydroxypolypoda-13,17,21-triene and 3-deoxyarabidiol ((5E)-6,10-dimethyl-2-[(3R,3aR,5aS,9aS,9bR)-3a,6,6,9a-tetramethyldodecahydro-1H-cyclopenta[a]naphthalen-3-yl]undeca-5,9-dien-2-ol), no formation of baciterpenol from tetraprenyl-beta-curcumene or onoceranoxide and 14beta-hydroxyonocera-8(26)-ene from 8alpha-hydroxypolypoda-13,17,21-triene, product ratios compared to wild-type, overview
Y167F
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site-directed mutagenesis, the enzyme mutant forms small quantity of tricyclic compounds, product ratios compared to wild-type, overview
Y167L
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site-directed mutagenesis, the enzyme mutant forms small quantity of tricyclic compounds, product ratios compared to wild-type, overview
Y167W
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site-directed mutagenesis, the enzyme mutant forms small quantity of tricyclic compounds, product ratios compared to wild-type, overview
additional information
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establishing of an (+)-ambrein production system by coexpressing the onoceroid synthase BmeTC from Bacillus megaterium with Saccharomyces cerevisiae squalene synthase (gene ScERG9) and Alicyclobacillus acidocaldarius squalene-hopene synthase mutant D377C (mutated gene shc) in Escherichia coli strain MEP43 (with deleted the beta-carotene synthesis module). The squalene synthase produces squalene, SHC enzyme mutant D377C produces 3-deoxyachilleol A from squalene, and BmTC forms ambrein from it, method optimization. Optimal temperature for ambrein production is 30-34°C
additional information
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for the biosynthesis of hydrophobic compounds such as terpenoids in Pichia pastoris, i.e.whole-cell production of (+)-ambrein, a central enzyme in the sterol biosynthesis pathway, squalene epoxidase Erg1, is targeted so that intracellular squalene levels in Pichia pastoris are strongly enhanced. Heterologous expression of AaSHC (EC 5.4.99.17) mutant D377C and enzyme BmeTC, and development of suitable methods to analyze all products of the engineered strain provide conclusive evidence of whole-cell (+)-ambrein production. Engineering of BmeTC leads to a remarkable one-enzyme system that is by far superior to the cascade, thereby increasing (+)-ambrein levels approximately 7fold in shake flask cultivation. Upscaling to 5 l bioreactor yields more than 100 mg/l of (+)-ambrein, demonstrating that metabolically engineered yeast Pichia pastoris represents a valuable, whole-cell system for high-level production of (+)-ambrein. Method evaluation and optimization, detailed overview
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Ueda, D.; Hoshino, T.; Sato, T.
Cyclization of squalene from both termini: identification of an onoceroid synthase and enzymatic synthesis of ambrein
J. Am. Chem. Soc.
135
18335-18338
2013
Priestia megaterium
brenda
Saga, Y.; Araki, T.; Araya, H.; Saito, K.; Yamazaki, M.; Suzuki, H.; Kushiro, T.
Identification of serratane synthase gene from the fern Lycopodium clavatum
Org. Lett.
19
496-499
2017
Lycopodium clavatum
brenda
Ke, D.; Caiyin, Q.; Zhao, F.; Liu, T.; Lu, W.
Heterologous biosynthesis of triterpenoid ambrein in engineered Escherichia coli
Biotechnol. Lett.
40
399-404
2018
Priestia megaterium
brenda
Tenkovskaia, L.; Murakami, M.; Okuno, K.; Ueda, D.; Sato, T.
Analysis of the catalytic mechanism of bifunctional triterpene/sesquarterpene cyclase Tyr167 functions to terminate cyclization of squalene at the bicyclic step
ChemBioChem
18
1910-1913
2017
Priestia megaterium
brenda
Ueda, D.; Hoshino, T.; Sato, T.
Cyclization of squalene from both termini Identification of an onoceroid synthase and enzymatic synthesis of ambrein
J. Am. Chem. Soc.
135
18335-18338
2013
Priestia megaterium
brenda
Moser, S.; Strohmeier, G.A.; Leitner, E.; Plocek, T.J.; Vanhessche, K.; Pichler, H.
Whole-cell (+)-ambrein production in the yeast Pichia pastoris
Metab. Eng. Commun.
7
e00077
2018
Priestia megaterium
brenda