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

Very-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.