Cofactor engineering for efficient production of α-farnesene by rational
modification of NADPH and ATP regeneration pathway in Pichia pastoris
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.