Trans β farnesene

S. cerevisiae ABC 276 was transformed with pRS315TEF-AtFS. Transformants were grown in SD media containing appropriate amino acids. Secondary culture was inoculated at 0.05 OD₆₀₀ and when OD₆₀₀ reaches to 0.6–0.8, culture was overlaid with 10% dodecane. After 48 h, the dodecane phase of the two- phase culture was collected by centrifugation of culture at 6000 rpm for 5 min. 1 μL of dodecane phase was subjected to GC-FID analysis. Samples were injected at a split ratio of 1:10. The oven temperature was initially held at 80 °C for 1 min and was increased at a rate of 10 °C/min to 250 °C where it was held for 1 min. Carrier gas was nitrogen. And the temperature of detector was maintained at 260 °C. All the conditions used for GC analysis was followed from (Wang et al., 2011 (link)). Standard curve of Trans β-Farnesene was prepared using GC- FID. Trans β-Farnesene (Cat. 73492) from Sigma Aldrich, India was used as standard.

At the end of the cultivation, the dodecane overlay was harvested by centrifugation at 4000 rpm for 3 min. Similarly, samples were collected during cultivations in bioreactors. Analysis was performed using a Focus GC-FID (Thermo Fisher Scientific) equipped with a ZB-50 column (Phenomenex, Torrance, CA, USA) as described previously with minor modifications [31 (link)]. The initial temperature was held at 50 °C for 1.5 min and then increased to 170 °C at a rate of 15 °C/min. After keeping the temperature constant for another 1.5 min, it was raised at the same rate to 300 °C and held for 3.0 min. The inlet temperature was set to 250 °C and 2 μL sample were injected in splitless mode. The base temperature of the flame was set to 300 °C. Farnesene was quantified using external calibration with trans-β-farnesene as analytical standard (≥90%, Sigma Aldrich). Samples were diluted in hexane and patchoulol (≥99%, a kind gift from Firmenich, Geneva, Switzerland) was added as internal standard. Concentrations of farnesene are stated based on the volume of the aqueous phase of the medium (mg/L_aq).