α-Farnesene, an acyclic volatile sesquiterpene, plays important roles in aircraft fuel, food flavoring, agriculture, pharmaceutical and chemical industries. Here, we enhanced α-farnesene production through reconstructing the biosynthetic pathways of NADPH and ATP in Pichia pastoris. First, the native oxiPPP was reconstructed by over-expressing the key enzymes in oxiPPP or/and inactivating glucose-6-phosphate isomerase (PGI), indicating that combined over-expression of ZWF1 and SOL3 improves NADPH supply and thus increasing α-farnesene production while inactivation of PGI was not because of the decreased cell growth. Next, different expression level of heterologous cPOS5 were introduced into P. pastoris, and found that low intensity expression of cPOS5 facilitated α-farnesene biosynthesis. Finally, ATP was increased by overexpression of APRT and inactivation of GPD1. The resultant strain P. pastoris X33-38 produced 3.09±0.37 g/L of α-farnesene in shake-flask fermentation, which was 41.7% higher than that of the parent strain. These results provide a new perspective to construct industrial-strength α-farnesene producer by rational modification of NADPH and ATP regeneration pathway in P. pastoris.
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