Innova 44 shaker

Measurements of SYN production were performed using a Duetz 96-well deep well system (Enzyscreen) coupled to an Innova 44 shaker (5 cm orbit) (New Brunswick Scientific) at 37°C and 300 rpm. HL1815 and HL1816 were used to measure SYN production (S1 Table). Seed cells were grown in LB in the presence of chloramphenicol for 4–5 h and thereafter grew in M9 overnight. Fresh M9 containing 200 mg/L OCT was inoculated with seed culture to 4%. These cells were transferred to a 96-well deep well plate, and each well contained 400 μl. 200 μl samples were withdrawn periodically for exometabolites analysis, while the remaining 200 μl cells were used to determine OD values using a SynergyMx microplate reader (BioTek). In vivo enzyme activity was averaged from four independent biological measurements normalized to dried cell weight. The conversion factor from OD to dried cell weight is 1 (i.e., 1 OD = 1 g/L) for our setup. It was observed that Pnmt activity was biomass dependent.

One litre MM consists of 50 ml l⁻¹ of salt solution (NaNO₃, 120 g l⁻¹; KCl, 10.4 g l⁻¹; MgSO_4·7H₂O, 10.4 g l⁻¹; KH₂PO₄, 30.4 g l⁻¹), 1 ml l⁻¹ trace elements (ZnSO₄·7H₂O, 2.2 g l⁻¹; H₃BO₃, 1.1 g l⁻¹; MnCl₂·4H₂O, 0.5 g l⁻¹; FeSO₄·7H₂O, 0.5 g l⁻¹; CoCl₂·5H₂O, 0.16 g l⁻¹; CuSO₄·5H₂O, 0.16 g l⁻¹; (NH₄)₆Mo₇O₂₄·4H₂O, 0.11 g l⁻¹; Na₄EDTA, 5 g l⁻¹), glucose, 10 g l⁻¹. Carbon-free MM does not contain glucose, nitrogen-free MM does not contain NaNO₃. 10 ml of this MM was inoculated with ~5 million spores, obtained from YPD agar plates, and grown for 2 days in MM or MM/0.5% (v v⁻¹) MetOH at 12 °C at 200 rpm, using an Innova 44 shaker (New Brunswick Scientific). Hyphae were induced by incubation in MM for 2–3 days, 200 rpm, at 18 or 27 °C, or at 18 °C in MM plus WLSE (500 µl WLSE stock solution per 100 ml of MM). WLSE stock solution was prepared by cutting and immersing 30 wheat leaves (12–14 days old and ~10 cm long; second leaf) in 15 ml of chloroform, ensuring that cut tissues did not contact the chloroform. After 3 min the solution was poured into a glass petri dish, and the solvent was left to evaporate overnight. The residue was re-suspended in methanol (final volume 2 ml = WLSE stock solution).

E. coli strains were cultivated on Luria–Bertani (LB) medium containing 100 mg/L ampicillin at 37°C. Yeast strains were maintained on YPD (10 g/L yeast extract, 20 g/L peptone, 20 g/L glucose). Yeast transformants were selected on YPD with 100 mg/L nourseothricin or SD‐Ura (20 g/L glucose, 6.7 g/L yeast nitrogen base, complete supplement mixture lacking uracil) plates. Solid plates contained 20 g/L agar.
Liquid media, including YPD, mineral/DELFT medium (pH 6.0; Jensen et al., 2014 (link)), and Feed‐In Time (FIT) medium (mineral medium where 20 g/L glucose is substituted with 60 g/L Enpresso® EnPump 200 substrate and 0.3% [v/v] Enpresso® Reagent A) were employed for yeast cell cultivation. Uracil was supplemented in a final concentration of 50 mg/L for strains lacking URA3.
Yeast cell cultivation in liquid medium was performed at 30°C throughout this study, in 96‐deep well plate at 300 rpm in a New Brunswick™ Innova® 44 shaker. Fresh single colonies were first pre‐cultured in 500 μl YPD or mineral medium overnight, and the resulting pre‐culture was sub‐inoculated into 600 μl mineral medium for sub‐cultivation. The mineral medium was preferentially used for pre‐culture, whereas YPD was used when the strains that have growth defects on the mineral medium were included. For the same batch cultivation, either mineral medium or YPD was used for the pre‐culture of all tested strains.

E. coli cells producing recombinant IacA, IacB and IacE proteins or their combinations were suspended in potassium phosphate buffer (10 mM, pH 7.7) supplemented with succinate (5 mM) to reach the 2× concentration of an initial culture and the IAA or derivative of IAA was added to a final concentration of 2 mM. Incubation of whole cells was performed with agitation (180 RPM, Innova44 Shaker, Eppendorf) at 30 °C overnight. Cells were removed by centrifugation at 16,000× g for 5 min and the supernatant was subjected to HPLS/MS analysis.