Fig. 3

Relative eGFP levels obtained with various elements of the GoldenPiCS toolbox. Expression strength of different promoters in comparison to P _GAP tested on different carbon sources (a, b), expression levels for P _GAP -controlled expression in combination with different transcriptional terminators (c), and comparison of P _GAP variants with alternative ‘-1’ nucleotides (d). At least 10 P. pastoris clones were screened to test promoter and terminator function in up to four different conditions: glycerol and glucose excess as present in batch cultivation (“G”, “D”), limiting glucose (“X”) and methanol feed (“M”), both representing fed batch. P _GAP to P _SHB17 were tested in ‘G’, ‘D’, ‘X’ and ‘M’ (A). P _TEF2 to P _PFK300 were validated in ‘D’, while P _GUT1 , P _THI11 and MUT-related promoters were tested in putative repressed and induced conditions (‘D’/‘G’, ‘D’+/−100 μM thiamine and ‘D’/M, respectively) (B). P _GAP variants with alternative ‘-1’ bases were analyzed in glucose excess (‘D’). Relative eGFP levels are related to P _GAP - controlled expression (A, B), terminator ScCYC1tt (B), or presented as relative value (C)