Journal: Metabolic engineering

Article Title: Engineering strategies for the fermentative production of plant alkaloids in yeast

doi: 10.1016/j.ymben.2015.05.001

Figure Lengend Snippet: Engineered pathway for sanguinarine biosynthesis in yeast from (R, S)-norlaudanosoline. Red arrows indicate reactions catalyzed by a plant cytochrome P450 enzyme. Structural class of the metabolites are indicated as green, protoberberine; blue, protopine; purple, benzophenanthridine. Ps6OMT, P. somniferum 6-O-methyltransferase; PsCNMT, P. somniferum coclaurine N-methyltransferase; Ps4’OMT, P. somniferum 4’-O-methyltransferase; PsBBE, P. somniferum berberine bridge enzyme; CPR, cytochrome P450 reductase; CFS, cheilanthifoline synthase; STS, stylopine synthase; TNMT, tetrahydroprotoberberine N-methyltransferase; MSH, cis-N-methylstylopine 14-hydroxylase; P6H, protopine 6-hydroxylase; DBOX, dihydrobenzophenanthridine oxidase.

Article Snippet: We generated standard curves for stylopine (ChromaDex), protopine (Sigma), and sanguinarine (Sigma) and used the most similar standard chemical structure to estimate concentration of intermediates for which no standard was available.

Techniques:

Journal: Metabolic engineering

Article Title: Engineering strategies for the fermentative production of plant alkaloids in yeast

doi: 10.1016/j.ymben.2015.05.001

Figure Lengend Snippet: Optimization of stylopine production through genetic and culture strategies. (A) Stylopine production in yeast strains as a function of the combination of the species variants of CFS and STS. CFS and STS variants were expressed from separate low-copy plasmids in CSY844. (B) Stylopine production in CSY904 grown under various culture conditions. 30 °C: 30 °C growth temperature; 25 °C: 25 °C growth temperature. 2 Stages: cultured at 25 °C with an initial growth phase followed by a production phase; Galactose: grown as described in 2 stages with 2% galactose used as the carbon source during the production phase. Metabolite production is determined by LC-MS analysis of culture media after indicated strains were grown for 96 h. Data is reported as the mean ± s.d. of at least 3 independent experiments.

Article Snippet: We generated standard curves for stylopine (ChromaDex), protopine (Sigma), and sanguinarine (Sigma) and used the most similar standard chemical structure to estimate concentration of intermediates for which no standard was available.

Techniques: Cell Culture, Liquid Chromatography with Mass Spectroscopy

Journal: Microbial Cell Factories

Article Title: Structure-function analysis of CYP719As involved in methylenedioxy bridge-formation in the biosynthesis of benzylisoquinoline alkaloids and its de novo production

doi: 10.1186/s12934-023-02024-2

Figure Lengend Snippet: Results of in vitro enzyme assay of CyCYP719As. The column graph indicated the conversion rate relative to the added substrate. A CyCYP719As catalyzed the conversion of ( S )-scoulerine 1 to ( S )-stylopine 3 . B CyCYP719As catalyzed the conversion of ( S )-tetrahydrocolumbamine 4 to ( S )-tetrahydroberberine 5 . Data reported are the means ± SD from triplicate analyses. ** indicates P < 0.01; nd, not detected

Article Snippet: The samples were immediately frozen in liquid nitrogen and stored at − 80 °C. ( S )-scoulerine, ( S )-stylopine, ( S )-cheilanthifoline, ( S )-nandinine, ( S )-tetrahydrocolumbamine, and ( S )-tetrahydroberberine with purity ≥ 95% were obtained from Shanghai Yuanye Bio-Technology.

Techniques: In Vitro, Enzymatic Assay

Journal: Microbial Cell Factories

Article Title: Structure-function analysis of CYP719As involved in methylenedioxy bridge-formation in the biosynthesis of benzylisoquinoline alkaloids and its de novo production

doi: 10.1186/s12934-023-02024-2

Figure Lengend Snippet: Conversion rates of CyCYP719As mutants relative to wild-type CyCYP719As. A Conversion rates of CyCYP719As mutants in catalyzing the D ring formation using ( S )-scoulerine as substrate to produce ( S )-cheilanthifoline. B Conversion rates of CyCYP719As mutants in catalyzing the A ring formation using ( S )-scoulerine as substrate to produce ( S )-nandinine. C Conversion rates of CyCYP719As mutants in catalyzing the A ring formation using ( S )-cheilanthifoline as substrate to produce ( S )-stylopine. D Conversion rates of CyCYP719As mutants in catalyzing the D ring formation using ( S )-nandinine as substrate to produce ( S )-stylopine. Data reported are the means ± SD from triplicate analyses. ** indicates P < 0.01; nd, not detected. The experimental data are shown in Additional file : Table S4

Article Snippet: The samples were immediately frozen in liquid nitrogen and stored at − 80 °C. ( S )-scoulerine, ( S )-stylopine, ( S )-cheilanthifoline, ( S )-nandinine, ( S )-tetrahydrocolumbamine, and ( S )-tetrahydroberberine with purity ≥ 95% were obtained from Shanghai Yuanye Bio-Technology.

Techniques:

Journal: Microbial Cell Factories

Article Title: Structure-function analysis of CYP719As involved in methylenedioxy bridge-formation in the biosynthesis of benzylisoquinoline alkaloids and its de novo production

doi: 10.1186/s12934-023-02024-2

Figure Lengend Snippet: UPLC-MS analysis of ( S )-stylopine produced by engineered S. cerevisiae strains. A The extracted ion chromatograms (EICs) of the ( S )-stylopine produced in yeast strain XJ0695 expression CyCYP719A39-CyCYP719A42 (pink) or CyCYP719A39 M120S -CyCYP719A42 (green). B The production of ( S )-stylopine in yeast strain XJ0695 expression CyCYP719A39-CyCYP719A42 (pink) or CyCYP719A39 M120S -CyCYP719A42 (green). Data reported are the means ± SD from triplicate analyses

Article Snippet: The samples were immediately frozen in liquid nitrogen and stored at − 80 °C. ( S )-scoulerine, ( S )-stylopine, ( S )-cheilanthifoline, ( S )-nandinine, ( S )-tetrahydrocolumbamine, and ( S )-tetrahydroberberine with purity ≥ 95% were obtained from Shanghai Yuanye Bio-Technology.

Techniques: Produced, Expressing