Cofactor Engineering for Efficient Production of a-Farnesene by Rational Modification of NADPH and ATP Regeneration Pathway in Pichia pastoris

alpha-Farnesene, an acyclic volatile sesquiterpene, plays important roles in aircraft fuel, food flavoring, agriculture, pharmaceutical and chemical industries. Here, by re-creating the NADPH and ATP biosynthetic pathways in Pichia pastoris, we increased the production of alpha-farnesene. First, the native oxiPPP was recreated by overexpressing its essential enzymes or by inactivating glucose-6-phosphate isomerase (PGI). This revealed that the combined over-expression of ZWF1 and SOL3 increases alpha-farnesene production by improving NADPH supply, whereas inactivating PGI did not do so because it caused a reduction in cell growth. The next step was to introduce heterologous cPOS5 at various expression levels into P. pastoris. It was discovered that a low intensity expression of cPOS5 aided in the production of alpha-farnesene. Finally, ATP was increased by the overexpression of APRT and inactivation of GPD1. The resultant strain P. pastoris X33-38 produced 3.09 +/- 0.37 g/L of alpha-farnesene in shake flask fermentation, which was 41.7% higher than that of the parent strain. These findings open a new avenue for the development of an industrial-strength alpha-farnesene producer by rationally modifying the NADPH and ATP regeneration pathways in P. pastoris.

Automated summary

By re-creating the NADPH and ATP biosynthetic pathways in Pichia pastoris, the production of alpha-farnesene was increased. Overexpressing ZWF1 and SOL3 improved NADPH supply, while inactivating PGI did not. Low intensity expression of cPOS5 aided in the production of alpha-farnesene. Finally, ATP production was increased by overexpressing APRT and inactivating GPD1. The resultant strain produced 41.7% more alpha-farnesene than the parent strain.