2.5. In vivo luciferase assay

The gene encoding luciferase (FLuc) was PCR amplified and subcloned into plasmid downstream of desired promoter cassette using restriction sites BamHI and XhoI. The designed plasmids encoding luciferase were transformed into yeast K. marxianus or S. cerevisiae. The 3–4 transformants were pooled and grown overnight into SD liquid growth media lacking uracil. The overnight grown primary culture was diluted to 0.05 O.D.₆₀₀ using 20 ​ml of sterile growth media (in 100 ​ml flask) with dextrose or xylose as carbon source. Cells were grown at either 30 ​°C or 45 ​°C at 200 ​rpm until cell O.D.₆₀₀ reached 0.8–1.0. The cells were harvested by centrifugation and re-suspended into 1 ​ml of YNB media. For measuring luciferase activity, 200 ​μl of 0.3 O.D.₆₀₀ ​cells were added with 50 ​μl of 1 ​mg/ml D-Luciferin (Sigma-Aldrich Cat #L9504), and the luminescence was measured in Multimode Plate Readers (TECAN Infinite M200 PRO, Switzerland).