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

Abstract

α-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 α-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 α-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 α-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 α-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 α-farnesene producer by rationally modifying the NADPH and ATP regeneration pathways in P. pastoris.

Creating NADP+‐Specific Formate Dehydrogenases from Komagataella phaffii by Enzymatic Engineering

“…At present, the primary approaches for the NADPH cofactor engineering in K. phaffii involve overexpressing glucose-6-phosphate dehydrogenase and 6- gluconolactonase. [34] We speculate that KphFDH/V19 can also serve as an endogenous enzyme for NADPH supply in K. phaffii.…”

Engineering Escherichia coli BL21 (DE3) for high‐yield production of germacrene A, a precursor of β‐elemene via combinatorial metabolic engineering strategies

“…Our result was consistent with recent studies that saw a 91% improvement in bisabolene production after zwf deletion (Zhang et al, 2021) and an enhancement in lycopene production after zwf inactivation (Zhou et al, 2013). The PPP is a crucial energy metabolism pathway, and the overexpression of zwf has been associated with increased NADPH supply for α‐farnesene synthesis (Chen et al, 2023). Hence, the inactivation of zwf could limit the availability of NADPH.…”

Biosynthesis Progress of High-Energy-Density Liquid Fuels Derived from Terpenes

“…In addition to NADPH, an adequate ATP supply is also crucial for increasing the production of terpenes. Chen et al overexpressed the endogenous adenine phosphoribosyltransferase (APRT), resulting in a 9.4% increase in intracellular ATP content, and also improved the yield of α-farnesene by 10.3% [142].…”