Extended Data Fig. 9 | Effect of extra gene copies on accumulation of TA
pathway intermediates and products in scopolamine-producing strain
CSY1296. a–f, An additional copy of each biosynthetic enzyme between
tropine and scopolamine was expressed from the following low-copy plasmids
in strain CSY1296: WfPPR, pCS4436 (a); AbUGT, pCS4440 (b); DsRed–AbLS,
pCS4526 (c); AbCYP80F1, pCS4438 (d); DsHDH, pCS4478 (e); DsH6H, pCS4439
(f); or a BFP control (pCS4208, pCS4212, or pCS4213). Transformed strains
were cultured in appropriate selective media at 25 °C for 96 h before
quantification of metabolites in the growth medium by LC–MS/MS analysis of
culture supernatant. Note that littorine accumulation was not observed for any
samples. Data indicate the mean of n = 3 biologically independent samples
(open circles) and error bars denote s.d. Metabolite titres are shown relative to
the BFP control with the same auxotrophic marker as the biosynthetic gene:
WfPPR (a), DsRed–AbLS (c), AbCYP80F1 (d), and DsH6H (f) relative to the
pCS4213 control (LEU2); AbUGT (b) relative to the pCS4208 control (URA3);
and DsHDH (e) relative to the pCS4212 control (TRP1). *P < 0.05, **P < 0.01,
***P < 0.001, Student’s two-tailed t-test. Statistical significance is shown
relative to corresponding controls. (a) PLA, P = 0.00950; PLA glucoside,
P = 3. 36 × 10−4; hyoscyamine, P = 0.00221; (b) tropine, P = 0.00500; PLA
glucoside, P = 3. 59 × 10−4; hyoscyamine, P = 0.0192; scopolamine, P = 6.90 × 10−4;
(c) PLA glucoside, P = 0.00544; hyoscyamine, P = 0.0165; scopolamine,
P = 3.43 × 10−4; (d) PLA, P = 0.0487; hyoscyamine, P = 0.0154; scopolamine,
P = 0.0159; (f) PLA, P = 0.0153; PLA glucoside, P = 0.0453; hyoscyamine,
P = 0.00958; scopolamine, P = 0.00816.