Fig. 6

Applying the library of BDPs helps to find the optimal expression condition for dual gene co-expression. For each of the three pair of genes tested, a different BDP performed best and the activity/yields for the same set of genes spanned a 5.2–50-fold range. a Highest taxadiene yields were achieved using a P_GAP-CAT1 fusion promoter for GGPPS and TDS co-expression. The designs based on different BDPs span a 50-fold range in yields. DAS2*-DAS1* denotes the improved promoter variant DAS2-d8-DAS1-d2d5 (Fig. 3c, Supplementary Fig. 2). Constitutive expression of the GGPPS gene was detrimental. Yields determined by GC-MS from shake flask cultivations (biological triplicates, mean value, and standard deviation shown) with a dodecane overlay. b Highest activity for the co-expression of human CYP2D6 and its associated CPR was achieved using the natural P_DAS1-DAS2 promoter in reverse orientation. The designs based on different BDPs span a 5.2-fold activity range. “2× AOX1 MDPs” indicates a control strain expressing the two genes using two monodirectional AOX1 promoters. The strains were pre-grown for 60 h on glucose and induced with methanol for 72 h. Activity of biological seven-fold replicates (mean value and standard deviation shown) was measured by a whole-cell bioconversion assay using 7-methoxy-4-(aminomethyl)-coumarin (MAMC) as substrate. c Bidirectional fusion promoters of P_CAT1 to P_AOX1 or P_GAP give the highest volumetric activities in the co-expression of secreted CalB and the chaperone PDI. The designs based on different BDPs span a 22-fold activity range. “CAT1, AOX1, MDPs” and “CAT1, GAP and MDPs” are control strains mimicking the best bidirectional designs with MDPs. Activities in the supernatant of biological quadruplicates (mean value and standard deviation shown) were measured after growth for 60 h on glucose and methanol induction for 72 h using a p-nitrophenyl butyrate (pNPB) assay