Deciphering the Biosynthetic Mechanism of Pelletierine in Lycopodium Alkaloid Biosynthesis

Abstract

Pelletierine, a proposed building block of Lycopodium alkaloids (LAs), was demonstrated to be synthesized via the non-enzymatic Mannich-like condensation of Δ 1 -piperideine and 3-oxoglutaric acid produced by two new type III PKSs (HsPKS4 and PcPKS1) characterized from Huperzia serrata and Phlegmariurus cryptomerianus, respectively. The findings provide new insights for further understanding the biosynthesis of LAs such as huperzine A.

A metabolic regulon reveals early and late acting enzymes in neuroactive Lycopodium alkaloid biosynthesis

“…Collectively, our results indicate that PtPIKS-1 and PtPIKS-2 are likely the key enzymes for forming 4PAA and pelletierine in Lycopodium alkaloid biosynthesis. This is in agreement with recent work that identified PIKS orthologs from two other Lycopodiaceae species with the same biosynthetic capacity (HsPKS4 from H. serrata, an ortholog of PtPIKS-2, 93% amino acid identity; PcPKS1 from Phlegmariurus cryptomerianus, an ortholog of PtPIKS-1, 97% amino acid identity) (32).…”

“…While no obvious products were observed when 1-piperideine was incubated alone as a substrate, addition of only malonyl-CoA to PtPIKS-1 led to accumulation of 3-oxoglutaric acid (Fig. 4 C and D), much like what was observed in analogous experiments with PYKS from Anisodus acutangulus (AaPYKS) (49), as well as the other described PIKS orthologs (32). The accumulation of 3-oxoglutaric acid suggests that PIKS is catalyzing the condensation of two malonyl-CoA units into a thioester-linked 3-oxoglutaryl moiety, and that this polyketide is either enzymatically or nonenzymatically hydrolyzed from the thioester linkage.…”

“…This past work has determined that Lys is processed into a heterocyclic imine (22)(23)(24), which is subsequently coupled to a polyketide substrate (25)(26)(27)(28), thereby generating the initial building blocks for Lycopodium alkaloid formation. Previous efforts have uncovered putative enzymes for these initial stages (29)(30)(31)(32), but our overall understanding of the Lycopodium alkaloid biosynthetic machinery was still largely incomplete. Here, we use a combination of long-read and short-read RNA sequencing (RNA-seq) to develop a robust transcriptomic data set for Phlegmariurus tetrastichus, a previously unsequenced club moss that produces HupA at high levels (33).…”

An Overview of the Medicinally Important Plant Type III PKS Derived Polyketides

“…Lycopodium alkaloids (LA) are classified as nitrogen-containing heterocyclic metabolites with diverse and stereochemically complex structures that have garnered attention due to their potent biological activities (Lichman, 2021 ). Some LA, such as huperzine A (HupA), acts as an inhibitor of acetylcholinesterase thereby are promising candidates for the treatment of Alzheimer's and myasthenia gravis disease in addition to harboring cytotoxic and neuroprotective activities (Ma and Gang, 2004 ; Wang et al, 2020 ). Classically, LAs have been categorized into four major groups (lycopodine, lycodine, fawcettimine, and phlegmarine) and since the discovery of the first LAs, lycopodine, around 400 LAs have been identified from the Lycopodiaceae and Huperziaceae families.…”

“…The biosynthesis mechanism of LA remained poorly understood in plants until now. In a recent report by Wang et al ( 2020 ), on the biosynthesis of pelletierine, two new 3-oxoglutaric acid synthesizers Type III PKSs, HsPKS4, and PcPKS1 of Huperzia serrata , and Phlegmariurus cryptomerianus , respectively, have been proposed to be involved in LA biosynthesis. HsPKS4 and PcPKS1 carry out the biosynthesis of pelletierine by providing 3-oxoglutaric acid which further undergoes a Mannich-like condensation with the Δ 1 -piperdeine starter substrate derived from lysine ( Figure 7G ).…”