Journal: bioRxiv

Article Title: Benzylisoquinoline alkaloid production in yeast via norlaudanosoline improves selectivity and yield

doi: 10.1101/2023.05.19.541502

Figure Lengend Snippet: (A) Biosynthesis of dihydrosanguinarine from ( S )-reticuline. (B) Stepwise construction of a de novo dihydrosanguinarine strain. Pathway enzymes were integrated into LN1015, a strain synthesizing ( S )-reticuline via ( S )-norlaudanosoline. Metabolites were extracted and, where possible, quantified by LC-MS. Error bars refer to mean and standard deviation of n=3 biological replicates. (C) Metabolite profile of intermediates between ( S )-scoulerine and ( S )- N - methylstylopine. Error bars refer to mean and standard deviation of n=3 biological replicates. (D) Fed-batch fermentation of LN1084, a strain synthesizing dihydrosanguinarine de novo . Samples were regularly collected for OD 600 and metabolite analysis. Two replicates are plotted with a best fit line to guide the eye. (E) Metabolite profile of target and intermediate compounds at the final time point of a representative fed-batch fermentation in panel (D). Concentrations of quantifiable metabolites are indicated in mg/L. Abbreviations: BBE, berberine bridge enzyme; CFS, ( S )- cheilanthifoline synthase; SPS, ( S )-stylopine synthase; TNMT, ( S )-tetrahydroprotoberberine N - methyltransferase; MSH, ( S )- N -methylstylopine hydroxylase; P6H, protopine 6-hydroxylase; DHS, dihydrosanguinarine; SN, sanguinarine. Symbols: ○ no enzyme; • one enzyme copy; •• two copies.

Article Snippet: Sources for authentic BIAs were: ( S )-norcoclaurine, TRC Inc. (North York, Ontario, Canada); ( S )-reticuline, gift from Dr. Peter Facchini; ( S )-scoulerine, ChromaDex (Irvine, Ca, USA); ( S )-stylopine, ChromaDex (Irvine, Ca, USA); protopine, TRC Inc.; sanguinarine, Sigma.

Techniques: Liquid Chromatography with Mass Spectroscopy, Standard Deviation

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