Orthogonal <i>in vivo</i> and <i>in vitro</i> membrane engineering enables human-like lipid remodeling of bacterial magnetosomes for functional TrkA display

The magnetotactic bacterium <i>Magnetospirillum magneticum</i> produces biogenic magnetic nanoparticles called magnetosomes, each comprising a magnetite core surrounded by a lipid bilayer. By expressing the human neurotrophin receptor TrkA, which is implicated in Alzheimer's disease, cancer, and other pathologies, on magnetosomes, we created a functional human transmembrane receptor platform that enables magnetic recovery and ligand screening. However, TrkA has limited activity within the native bacterial membrane, likely due to the absence of key eukaryotic lipids such as phosphatidylcholine (PC) and cholesterol (Chol). To overcome this limitation, we combined <i>in vivo</i> and <i>in vitro</i> membrane engineering to remodel the magnetosome envelope and closely mimic the human cell membrane. Specifically, PC was biosynthesized <i>in vivo</i> by co-expressing phosphatidylcholine synthase, and purified magnetosomes were subsequently enriched <i>in vitro</i> with Chol using methyl-β-cyclodextrin. This dual-lipid strategy significantly enhanced TrkA-ligand-binding and autophosphorylation. These findings show that orthogonal membrane engineering can precisely tailor the lipid composition of bacterial magnetosomes to recreate mammalian-like bilayers, rescuing the function of complex human receptors in a microbial environment. This approach expands the metabolic and synthetic biology toolkit for producing functional membrane proteins on scalable magnetic supports and establishes a cost-effective platform for the discovery of TrkA and other membrane receptors.IMPORTANCEMembrane receptors drive essential signaling in health and disease; however, they are difficult to study and screen because most platforms fail to reproduce human-like membranes at scale. Magnetosomes from the bacterium <i>Magnetospirillum magneticum</i> AMB-1 offer a simple alternative: lipid-bounded, magnetic nanoparticles that can be purified in one step. This work establishes human-like remodeling of magnetosome membranes by combining <i>in vivo</i> phosphatidylcholine synthesis with the first <i>in vitro</i> cholesterol loading of these particles. Displaying human tropomyosin receptor kinase A on the remodeled membranes preserved and enhanced the receptor function without complex purification or reconstitution. Because magnetosomes can be produced inexpensively and recovered magnetically, this approach enables practical, high-throughput assays for ligand discovery and inhibitor testing. The strategy is broadly applicable to other human membrane proteins, linking microbial biotechnology with human membrane biology to accelerate translational research.