Conclusions
α-Farnesene is biosynthesized from farnesyl pyrophosphate (FPP) and dimethylallyl pyrophosphate (DMAPP), which are mainly produced through the mevalonate (MVA) pathway in P. pastoris 3, 6, and thus 6 molecules of NADPH and 9 molecules of ATP are need to produce 1 molecule of α-farnesene (Fig. 1). Although many effective strategies have been used to increase the α-farnesene production in yeast, for example, in P. pastoris 3,Saccharomyces cerevisiae 51-52 andYarrowia lipolytica 53-54, these studies mainly focused on modifying the carbon’s metabolism pathway to enhance the carbon flux in α-farnesene biosynthetic pathway. Here, we first tried to rationally reconstruct the biosynthetic pathways of NADPH and ATP to further increase the α-farnesene production in a α-farnesene high-producing strain P. pastoris X33-30*. The resultant strainP. pastoris X33-38 produced 3.09±0.37 g/L of α-farnesene after 72 h in shake-flask fermentation, which is the highest value ever reported as we know it (Table 4).
In P. pastoris , oxiPPP is the main pathway for NADPH generation, but overexpression of the all genes in oxiPPP is not a brilliant choice for increasing α-farnesene production because of the disturbance of the carbon flux in the PPP26 and acetyl-CoA biosynthetic pathway31. Combined over-expression of ZWF1 and SOL3 improves the NADPH supply and thus increasing α-farnesene production. Moreover, the intracellular NADPH level can be further increased by heterologous expression of cPOS5 rather than inactivation of PGI, and thus the α-farnesene production reaches to 2.77±0.18 g/L in strain X33-35. As the other key cofactor in α-farnesene production, increasing the ATP supply also plays an important role in promoting α-farnesene production. The strain X33-38 with overexpression of APRT and deletion of NADH-dependent dihydroxyacetone phosphate reductase. To increase the supply of AMP and NADH for ATP generation shows the obvious increased cell growth and α-farnesene production. Therefore, rational modification of NADPH and ATP regeneration pathway plays a vital role in facilitating α-farnesene biosynthesis in P. pastoris . These results also provide a new direction and reference to construct NADPH- and/or ATP-dependent high value-added products producing strains.