A cytochrome P450 monooxygenase responsible for the C-22 hydroxylation step in the Paris polyphylla steroidal saponin biosynthesis pathway

Yin, Yong; Gao, Linhui; Zhang, Xianan; Gao, Wei

doi:10.1016/j.phytochem.2018.09.005

Cited by 34 publications

(30 citation statements)

References 31 publications

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“…The ion source temperature was 250 °C and the ionization voltage was 70 eV. The temperature program was as follows: after keeping the column temperature at 180 °C for 1 min, the temperature was raised to 280 °C at a rate of 20 °C min -1 , then to 300 °C at a rate of 2 °C min -1 with a 2 min hold (Yin et al 2018).…”

Section: Determination Of the Enzymatic Product By Gc-msmentioning

confidence: 99%

Cloning and functional characterization of the β-amyrin synthase genefrom Bupleurum chinense

Li¹,

Wang²,

Qi³

et al. 2020

Biol. plant.

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Bupleurum chinense DC., a source of the traditional Chinese medicine Bupleuri Radix, is rich in triterpenoid saikosaponins with high pharmacological activities. The enzyme β-amyrin synthase (BAS), which converts the precursor 2,3-oxidosqualene to produce the triterpene skeleton, is crucial for the biosynthesis of triterpenoid saponins. In this study, we cloned the full-length sequence of the BAS gene from B. chinense, conducted a bioinformatics analysis, and expressed it in Saccharomyces cerevisiae to investigate its function. The cDNA of β-amyrin synthase (BcBAS, GenBank accession number: MN186093) cloned from aseptic seedlings of B. chinense was 2 307 bp with a 2 286 bp open reading frame coding for 761 amino acids. Phylogenetic analysis suggests that the BcBAS protein was closely related to the BAS proteins from Panax ginseng and Betula platyphylla as chromatography mass spectrometry analysis showed that the enzymatic product was indeed β-amyrin, the precursor of oleanane type triterpenes. Overall, our findings lay the foundation for in-depth analysis of the biosynthesis pathway of saikosaponins.

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Section: Determination Of the Enzymatic Product By Gc-msmentioning

confidence: 99%

Cloning and functional characterization of the β-amyrin synthase genefrom Bupleurum chinense

Li¹,

Wang²,

Qi³

et al. 2020

Biol. plant.

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“…CYP90B27 was identified from functional transcriptome analysis, and the heterologous expression of CYP90B27 led to a descent conversion towards 22(R)-hydroxycholesterol, which confirmed CYP90B27 as the 22α-hydroxylase (Fig. 3, Table 2) [64]. More recently in 2019, RH6829 was utilized in the elucidation of the biosynthesis of diosgenin, the aglycone of a class of spirostanol-type saponins that is synthesized from cholesterol [65].…”

Section: Synthesis Of Steroids Without C24-alkylmentioning

confidence: 93%

“…S. cerevisiae has been extensively studied and utilized for genetic and metabolic engineering, with the well-established genetic modification tools [87]. Therefore, it is widely used in enzyme characterizations in addition to bioproduction purpose, such as the functional identification of CYP90B27 [64] and 24ISO [79]. Y. lipolytica is an industrial oleaginous strain and is usually used for high-yield lipid production, up to 70% w/w dry cell weight [88].…”

Section: Future Perspectivementioning

confidence: 99%

“…The cholesterol-producing S. cerevisiae strain RH6829 is, thus, often used for enzyme functional characterization to elucidate the biosynthesis of cholesterol-derived phytochemicals. One recent example is the characterization of the 22 R -hydroxylase CYP90B27 involved in the synthesis of polyphyllins in the medicinal herb Paris polyphylla [ 64 ]. The first step from cholesterol to saponins and steroidal alkaloids is believed to be C22-hydroxylation and catalyzed by a cytochrome P450.…”

Section: Synthesis Of Steroids Without C24-alkylmentioning

confidence: 99%

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Yeast as a promising heterologous host for steroid bioproduction

2020

Journal of Industrial Microbiology and Biotechnology

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With the rapid development of synthetic biology and metabolic engineering technologies, yeast has been generally considered as promising hosts for the bioproduction of secondary metabolites. Sterols are essential components of cell membrane, and are the precursors for the biosynthesis of steroid hormones, signaling molecules, and defense molecules in the higher eukaryotes, which are of pharmaceutical and agricultural significance. In this mini-review, we summarize the recent engineering efforts of using yeast to synthesize various steroids, and discuss the structural diversity that the current steroidproducing yeast can achieve, the challenge and the potential of using yeast as the bioproduction platform of various steroids from higher eukaryotes.

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“…For instance, the desmosterolproducing yeast and 24-methylenecholesterol-producing yeast constructed through expressing DWF5 into erg6 and erg4/erg5 inactivated strains, respectively, have been utilized to characterize sterol Δ 24(25) reductase involved in cholesterol biosynthesis 25 , novel sterol Δ 24 (28) reductase involved in campesterol production 26 , and Δ 24 isomerase putatively used in withanolide biosynthesis 27 . The cholesterol-producing yeast constructed through expressing 24dehydrocholesterol reductase (DHCR24) and DHCR7 in the erg5/erg6 inactivated yeast strain has been utilized to confirm the function of CYP90B27 in synthesizing 22(R)-hydroxycholesterol from cholesterol 28 . The same cholesterol-producing yeast strain has also been utilized to reconstruct diosgenin biosynthesis with the expression of PpCYP90G4/PpCYP94D108 or TfCYP90B50/TfCYP82J17.…”

Section: Introductionmentioning

confidence: 99%

Engineering of Phytosterol-Producing Yeast Platforms for Functional Reconstitution of Downstream Biosynthetic Pathways

Xu¹,

Chen²,

Li³

2020

Preprint

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As essential structural molecules for plant plasma membranes, phytosterols are key intermediates for the synthesis of many downstream specialized metabolites of pharmaceutical or agricultural significance, such as brassinosteroids and withanolides. Saccharomyces cerevisiae has been widely used as an alternative producer for plant secondary metabolites. Establishment of heterologous sterol pathways in yeast, however, has been challenging due to either low efficiency or structural diversity, likely a result of crosstalk between the heterologous phytosterol and the endogenous ergosterol biosynthesis. For example, in this study, we engineered campesterol production in yeast using plant enzymes; although we were able to enhance the titer of campesterol to ~40mg/L by upregulating the mevalonate pathway, no conversion to downstream products was detected upon the introduction of downstream plant enzymes. Further investigations uncovered two interesting observations about sterol engineering in yeast. First, many heterologous sterols tend to be efficiently and intensively esterified in yeast, which drastically impedes the function of downstream enzymes. Second, yeast can overcome the growth deficiency caused by altered sterol metabolism through repeated culture. By employing metabolic engineering, strain evolution, fermentation engineering, and pathway reconstitution, we were able to establish a set of phytosterol-producing yeast strains with decent growth and titer of campesterol (~ 7mg/L), βsitosterol (~2mg/L), 22-hydroxycampesterol (~1mg/L), and 22-hydroxycampest-4-en-3-one (~4mg/L). This work resolves the technical bottlenecks in phytosterol-derived pathway reconstitution in the backer's yeast and opens up opportunities for efficient bioproduction and pathway elucidation of this group of phytochemicals.

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A cytochrome P450 monooxygenase responsible for the C-22 hydroxylation step in the Paris polyphylla steroidal saponin biosynthesis pathway

Cited by 34 publications

References 31 publications

Cloning and functional characterization of the β-amyrin synthase genefrom Bupleurum chinense

Cloning and functional characterization of the β-amyrin synthase genefrom Bupleurum chinense

Yeast as a promising heterologous host for steroid bioproduction

Engineering of Phytosterol-Producing Yeast Platforms for Functional Reconstitution of Downstream Biosynthetic Pathways

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