Verification of the phenylpropanoid pinoresinol biosynthetic pathway and its glycosides in Phomopsis sp. XP-8 using ¹³C stable isotope labeling and liquid chromatography coupled with time-of-flight mass spectrometry

Phomopsis sp. XP-8, an endophytic fungus from the bark of Tu-Chung (EucommiaulmoidesOliv), revealed the pinoresinol diglucoside (PDG) biosynthetic pathway after precursor feeding measurements and genomic annotation. To verify the pathway more accurately, [¹³C₆]-labeled glucose and [¹³C₆]-labeled phenylalanine were separately fed to the strain as sole substrates and [¹³C₆]-labeled products were detected by ultra-high performance liquid chromatography-quantitative time of flight mass spectrometry. As results, [¹³C₆]-labeled phenylalanine was found as [¹³C₆]-cinnamylic acid and p-coumaric acid, and [¹³C₁₂]-labeled pinoresinol revealed that the pinoresinol benzene ring came from phenylalanine via the phenylpropane pathway. [¹³C₆]-Labeled cinnamylic acid and p-coumaric acid, [¹³C₁₂]-labeled pinoresinol, [¹³C₁₈]-labeled pinoresinol monoglucoside (PMG), and [¹³C₁₈]-labeled PDG products were found when [¹³C₆]-labeled glucose was used, demonstrating that the benzene ring and glucoside of PDG originated from glucose. It was also determined that PMG was not the direct precursor of PDG in the biosynthetic pathway. The study verified the occurrence of the plant-like phenylalanine and lignan biosynthetic pathway in fungi.