Figure captions
Figure 1 - The schematic diagram of α-farnesene biosynthetic pathways with NADPH and ATP regeneration pathway in Pichia pastoris. The pathways of NADPH biosynthesis are shown in blue lines, and the pathways of NADPH catabolism are shown in red lines. The pathways of ATP biosynthesis are shown in green lines, and the pathways of ATP catabolism are shown in pink lines. The key genes were listed in ellipses. Abbreviations: IPP (isopentenyl pyrophosphate), DMAPP (dimethylallyl pyrophosphate), GPP (geranyl pyrophosphate), FPP (farnesyl pyrophosphate).
Figure 2 – Screening the best enzyme in oxiPPP for α-farnesene biosynthesis. (a) Overexpression of the key enzymes in oxiPPP affects the intracellular NADPH level. (b) The effects of overexpression of the key enzymes in oxiPPP on α-farnesene production after 72 h cultivation. The strain X33-30* was used as the control (Grey bar), and the best strain is shown in red bar. These data represent average values and standard deviations achieved from three independent experiments.
Figure 3 – Optimization of the expression combination ways of the enzymes in oxiPPP for α-farnesene biosynthesis. (a) The effects of the different expression combination ways on the intracellular NADPH level. (b) The effects of the different expression combination ways on α-farnesene production after 72 h cultivation. The strain X33-30*S was used as the control (Grey bar), and the best strain is shown in red bar. These data represent average values and standard deviations achieved from three independent experiments.
Figure 4 – Inactivation of PGI1 negatively affects α-farnesene biosynthesis. (a) α-Farnesene titers. (b) Dry cell weight (DCW). (c) The intracellular NADPH level at 24 h and 72 h. The strains X33-30* and X33-31 were used as the control (Grey bar). These data represent average values and standard deviations achieved from three independent experiments.
Figure 5 - Optimization of the expression level of cPOS5 for α-farnesene biosynthesis. (a) Dry cell weight (DCW). (b) The intracellular NADPH level at 24 h and 72 h. (c) α-Farnesene titers and productivity. The strain X33-31 was used as the control (Grey bar). These data represent average values and standard deviations achieved from three independent experiments.
Figure 6 –Overexpression of APRT to increase ATP supply for α-farnesene production. (a) The cell growth of strain X33-37 with overexpression of APRT. (b) The α-farnesene production of strain X33-37 with overexpression of APRT. The strain X33-35 without overexpression of APRT was used as the control. These data represent average values and standard deviations achieved from three independent experiments.
Figure 7 – Inactivation of GPD1 to decrease the NADH consumption in shunt pathway. (a) α-farnesene titers of strains X33-37 and X33-38. (b) DCW of strains X33-37 and X33-38. (c) Glycerol titers of strains X33-37 and X33-38. These data represent average values and standard deviations achieved from three independent experiments.
Table 1. The main strains used in the study