Pathway elucidation and heterologous reconstitution of the long‐chain alkane pentadecane biosynthesis from Pogostemon cablin

Abstract

SummaryVery‐long‐chain (VLC) alkanes are major components of hydrophobic cuticular waxes that cover the aerial epidermis of land plants, serving as a waterproofing barrier to protect the plant against environmental stresses. The mechanism of VLC‐alkane biosynthesis has been extensively elucidated in plants. However, little is known about the biosynthesis of long‐chain alkanes (LC, C13 ~ C19) such as pentadecane in plants. Alkanes with different chain lengths are also major constituents of fossil fuels and thus the discovery of the alkane biosynthetic machinery in plants would provide a toolbox of enzymes for the production of renewable hydrocarbon sources and next generations of biofuels. The top leaves of Pogostemon cablin at young stage accumulate large amounts of LC‐alkane pentadecane, making this plant an excellent system for the elucidation of LC‐alkane biosynthetic machinery in plant. We show here that LC‐alkane pentadecane biosynthesis in P. cablin involves an endoplasmic reticulum (ER)‐localized complex made of PcCER1‐LIKE3 and PcCER3, homologues of Arabidopsis ECERIFERUM1 (AtCER1) and AtCER3 proteins that are involved in Arabidopsis VLC‐alkane biosynthesis. We reconstitute the biosynthesis of pentadecane in Nicotiana benthamiana by co‐expression of PcCER1‐LIKE3 and PcCER3 and further improve its production by silencing multifunctional acetyl‐CoA carboxylases involved in fatty acid elongation pathway. Taken together, we uncovered the key biosynthetic machinery of LC‐alkane pentadecane in P. cablin and demonstrated that using these newly identified enzymes to engineer this LC‐alkane for liquid biofuel production in a heterologous plant host is possible.