Many opportunistic bacteria that infect the upper respiratory tract have phosphorylcholine (ChoP) integrated into their surface structures, which contributes to host mimicry and promotes colonisation and survival inside host cells. ChoP modification is not confined to carbohydrates; ChoP is now known to be decorated on surface-exposed proteins and bacterial virulence factors. In Neisseria meningitidis, pili are major virulence factors and are post-translationally modified by ChoP. PptA, phosphorylcholine transferase A recognize the substrate sequence 153CRDASDAS160, present within the C-terminus of pilin and adds ChoP to Ser157 and Ser160. ChoP on pilin is crucial for the early colonization of the airway epithelium as it mediates adherence via binding to the platelet activating factor receptor expressing on airway epithelial cells. We previously found that, unlike bacteria that possess ChoP-linked glycoconjugates, N. meningitidis does not contain any of the characterized ChoP biosynthetic pathways, therefore the pathway(s) required for ChoP production prior to pilin post-translational modification is still unknown. In this study, we aim to investigate the donor molecules for ChoP synthesis in N. meningitidis. Here, we used biochemical approaches such as surface plasmon resonance (SPR) screening of ChoP donor molecules candidates and Neisseria cell lysate to identify binding to PptA, thereby identifying candidate donor molecules. We isolated the soluble, catalytic domain of PptA and demonstrated that it binds to the target peptide receptor for ChoP, 53CRDASDAS160. Of the candidate donor molecules tested for binding to PptA, phosphatidylcholine bound with the highest affinity (KD [equilibrium dissociation constant]. 377±53.6nM). This finding suggests that phosphatidylcholine may be the donor molecule for ChoP conjugate synthesis and PptA is responsible for transferring ChoP from phosphatidylcholine to the C-terminus of pilin. In the absence of the choline source and the expression of pilin and PptA, the cell lysate components from N. meningitidis demonstrate a high binding affinity. These findings demonstrate that N. meningitidis has a novel ChoP biosynthetic pathway.