Analysis of the Catalytic Mechanism of Bifunctional Triterpene/Sesquarterpene Cyclase: Tyr167 Functions To Terminate Cyclization of Squalene at the Bicyclic Step

Tenkovskaia, Liudmila; Murakami, Mizuki; Okuno, Kotone; Ueda, Daijiro; Sato, Tsutomu

doi:10.1002/cbic.201700329

Cited by 7 publications

(14 citation statements)

References 22 publications

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“…Thereby, oxidosqualene cyclases are not able to protonate a carbon-carbon double bond ( Gao et al, 2012 ). Initial studies on the catalytic mechanism of Bme TC applying homology modelling and subsequent testing of active site variants have been published very recently ( Tenkovskaia et al, 2017 ). It was shown that despite a high similarity of the active site architecture between Aa SHC and Bme TC, Bme TC exhibits a, so far, unique catalytic mechanism.…”

Section: Resultsmentioning

confidence: 99%

Whole-cell (+)-ambrein production in the yeast Pichia pastoris

Moser

Strohmeier

Leitner

et al. 2018

Metabolic Engineering Communications

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The triterpenoid (+)-ambrein is a natural precursor for (-)-ambrox, which constitutes one of the most sought-after fragrances and fixatives for the perfume industry. (+)-Ambrein is a major component of ambergris, an intestinal excretion of sperm whales that is found only serendipitously. Thus, the demand for (-)-ambrox is currently mainly met by chemical synthesis. A recent study described for the first time the applicability of an enzyme cascade consisting of two terpene cyclases, namely squalene-hopene cyclase from Alicyclobacillus acidocaldarius (AaSHC D377C) and tetraprenyl-β-curcumene cyclase from Bacillus megaterium (BmeTC) for in vitro (+)-ambrein production starting from squalene. Yeasts, such as Pichia pastoris, are natural producers of squalene and have already been shown in the past to be excellent hosts for the biosynthesis of hydrophobic compounds such as terpenoids. By targeting a central enzyme in the sterol biosynthesis pathway, squalene epoxidase Erg1, intracellular squalene levels in P. pastoris could be strongly enhanced. Heterologous expression of AaSHC D377C and BmeTC and, particularly, development of suitable methods to analyze all products of the engineered strain provided conclusive evidence of whole-cell (+)-ambrein production. Engineering of BmeTC led to a remarkable one-enzyme system that was by far superior to the cascade, thereby increasing (+)-ambrein levels approximately 7-fold in shake flask cultivation. Finally, upscaling to 5 L bioreactor yielded more than 100 mg L−1 of (+)-ambrein, demonstrating that metabolically engineered yeast P. pastoris represents a valuable, whole-cell system for high-level production of (+)-ambrein.

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Section: Resultsmentioning

confidence: 99%

Whole-cell (+)-ambrein production in the yeast Pichia pastoris

Moser

Strohmeier

Leitner

et al. 2018

Metabolic Engineering Communications

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“…2 and 3). Whereas the product of BmeTC Y167A containing a novel tricyclic compound was previously reported 20 , the enzyme activity was first revealed in this study.…”

Section: Resultsmentioning

confidence: 61%

“…E. coli BL21(DE3) harboring pColdTF-BmeTC D373C/L596A was grown at 37 °C in LB medium (1 L) with 100 μg mL -1 ampicillin 20 . Expression of the recombinant protein was induced by adding 0.1 mM IPTG when OD 600 reached ~ 0.6 20 . Further cultivation of BL21(DE3) recombinants was performed for 24 h at 15°C 20 .…”

Section: Methodsmentioning

confidence: 99%

“…Expression of the recombinant protein was induced by adding 0.1 mM IPTG when OD 600 reached ~ 0.6 20 . Further cultivation of BL21(DE3) recombinants was performed for 24 h at 15°C 20 . E. coli cells expressing recombinant BmeTC D373C/L596A were harvested by centrifugation and resuspended in buffer A (15 mL/5 g) containing 50 mM Tris-HCl (pH 7.5), 2.5 mM dithiothreitol, 1 mM EDTA, 0.1% ascorbic acid and 0.1% Tween-80 20 .…”

Section: Methodsmentioning

confidence: 99%

“…Further cultivation of BL21(DE3) recombinants was performed for 24 h at 15°C 20 . E. coli cells expressing recombinant BmeTC D373C/L596A were harvested by centrifugation and resuspended in buffer A (15 mL/5 g) containing 50 mM Tris-HCl (pH 7.5), 2.5 mM dithiothreitol, 1 mM EDTA, 0.1% ascorbic acid and 0.1% Tween-80 20 . Cells were disrupted by sonication with UP200s (Hielscher Ultrasonics GmbH) at 4-10 °C for 15 min 20 .…”

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Construction of an artificial system for ambrein biosynthesis and investigation of some biological activities of ambrein

Yamabe¹,

Kawagoe²,

Okuno³

et al. 2020

Sci Rep

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Ambergris, a sperm whale metabolite, has long been used as a fragrance and traditional medication, but it is now rarely available. The odor components of ambergris result from the photooxidative degradation of the major component, ambrein. The pharmacological activities of ambergris have also been attributed to ambrein. However, efficient production of ambrein and odor compounds has not been achieved. Here, we constructed a system for the synthesis of ambrein and odor components. First, we created a new triterpene synthase, “ambrein synthase,” for mass production of ambrein by redesigning a bacterial enzyme. The ambrein yields were approximately 20 times greater than those reported previously. Next, an efficient photooxidative conversion system from ambrein to a range of volatiles of ambergris was established. The yield of volatiles was 8–15%. Finally, two biological activities, promotion of osteoclast differentiation and prevention of amyloid β-induced apoptosis, were discovered using the synthesized ambrein.

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A Non‐Enzymatic Pathway with Superoxide in Intracellular Terpenoid Synthesis

et al. 2018

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Non-C 5 -units terpenoids (norisoprenoids) with an acetonyl group are widely distributed in nature.H owever, studies on the biosynthesis of norisoprenoids are scarce.Now, the C 33 norisoprenoid, (all-E)-farnesylfarnesylacetone,w as identified from Bacillus spp.and it was elucidated for the first time that superoxidem ediates the cleavage of menaquinones (vitamin K) to form norisoprenoids in saponification treatment. From in vivo experiments using gene-disrupted Bacillus subtilis strains targeted for enzymes responsible for menaquinone biosynthesis and for superoxided ismutase,i tw as suggested that the non-enzymatic cleavage (autoxidation) of menaquinone with superoxide resulted in norisoprenoid synthesis in Bacillus cells.F urthermore,t he bioactive norisoprenoids,farnesylacetone and phytone,were produced in Bacillus cells by this novel synthesis system.

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Analysis of the Catalytic Mechanism of Bifunctional Triterpene/Sesquarterpene Cyclase: Tyr167 Functions To Terminate Cyclization of Squalene at the Bicyclic Step

Cited by 7 publications

References 22 publications

Whole-cell (+)-ambrein production in the yeast Pichia pastoris

Whole-cell (+)-ambrein production in the yeast Pichia pastoris

Construction of an artificial system for ambrein biosynthesis and investigation of some biological activities of ambrein

A Non‐Enzymatic Pathway with Superoxide in Intracellular Terpenoid Synthesis

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