Due to their highly regulated biosynthesis, magnetosomes biomineralized by magnetotactic bacteria represent natural magnetic nanoparticles with unique physical and chemical properties. They consist of a magnetite core that is surrounded by a biological membrane and are therefore reminiscent to magnetic “core–shell” nanoparticles. Their usability in many nanotechnological and biomedical applications would be further improved by the introduction of additional catalytic and imaging modalities. Here, a new in vivo strategy is explored for magnetosome display of foreign polypeptides with maximized protein‐to‐particle ratios. Arrays of up to five monomers of the model enzyme glucuronidase GusA plus the additional fluorophore mEGFP are genetically fused as single large hybrid proteins to highly expressed magnetosome protein anchors. In total, about 190 GusA monomers are covalently attached to individual particles. Assuming layers of GusA rows surrounding the particles, the monomers would thus cover up to 90% of the magnetosome surface. The approach generates nanoparticles that exhibit magnetism, fluorescence, and stable catalytic activities, which are stepwise increased with the number of GusA monomers. In summary, multicopy expression of arrayed foreign proteins represents a powerful methodology for the biosynthesis of tailored biohybrid magnetic nanoparticles with several genetically encoded and tunable functionalities.

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Their unique material characteristics, i.e. high crystallinity, strong magnetization, uniform shape and size, and the ability to engineer the enveloping membrane in vivo make bacterial magnetosomes highly interesting for many biomedical and biotechnological applications. In this study, a versatile toolkit is developed for the multifunctionalization of magnetic nanoparticles in the magnetotactic bacterium Magnetospirillum gryphiswaldense, and the use of several abundant magnetosome membrane proteins as anchors for functional moieties is explored. High‐level magnetosome display of cargo proteins enables the generation of engineered nanoparticles with several genetically encoded functionalities, including a core–shell structure, magnetization, two different catalytic activities, fluorescence and the presence of a versatile connector that allows the incorporation into a hydrogel‐based matrix by specific coupling reactions. The resulting reusable magnetic composite demonstrates the high potential of synthetic biology for the production of multifunctional nanomaterials, turning the magnetosome surface into a platform for specific versatile display of functional moieties.

Magnetosomes are magnetic nanoparticles biomineralized by magnetotactic bacteria. They consist of a monocrystalline magnetite core enveloped by the magnetosome membrane, which harbors a set of specialized proteins. For the alphaproteobacterium Magnetospirillum gryphiswaldense genetic techniques were developed for engineering both crystal morphology and the enveloping membrane, thereby generating building blocks for magnetic organic–inorganic hybrid materials. Genetic manipulation of magnetite biomineralization enabled the generation of core-engineered nanoparticles with adjusted magnetic and physicochemical properties. Functionalization of the particle surface was achieved by genetic expression of enzymes and peptides genetically fused to abundant magnetosome anchor proteins. High-level expression allowed the generation of multifunctional nanoparticles with maximized protein-to-particle ratios. This allowed for the tuning of surface properties (charge and hydrodynamic diameter), and the colloidal and enzymatic stability was improved by coating with inorganic and organic shells. The expression of molecular connectors might serve as scaffolds for the introduction of further functionalities. Overall, this demonstrates that the ‘synthetic biology’ approach enables the generation of multifunctional, magnetic hybrid materials with a tuned property spectrum exceeding those of conventional materials, and the combination of different biogenic materials generates fully genetically encoded biocomposites with enhanced potential for various biotechnological and biomedical applications.

Magnetotactic bacteria (MTB) stand out by their ability to manufacture membrane-enclosed magnetic organelles, so-called magnetosomes. Previously, it has been assumed that a genomic region of approximately 100 kbp, the magnetosome island (MAI), harbors all genetic determinants required for this intricate biosynthesis process. Recent evidence, however, argues for the involvement of additional auxiliary genes that have not been identified yet. In the present study, we set out to delineate the full gene complement required for magnetosome production in the alphaproteobacterium Magnetospirillum gryphiswaldense using a systematic genome-wide transposon mutagenesis approach. By an optimized procedure, a Tn5 insertion library of 80,000 clones was generated and screened, yielding close to 200 insertants with mild to severe impairment of magnetosome biosynthesis. Approximately 50% of all Tn5 insertion sites mapped within the MAI, mostly leading to a nonmagnetic phenotype. In contrast, in the majority of weakly magnetic Tn5 insertion mutants, genes outside the MAI were affected, which typically caused lower numbers of magnetite crystals with partly aberrant morphology, occasionally combined with deviant intracellular localization. While some of the Tn5-struck genes outside the MAI belong to pathways that have been linked to magnetosome formation before (e.g., aerobic and anaerobic respiration), the majority of affected genes are involved in so far unsuspected cellular processes, such as sulfate assimilation, oxidative protein folding, and cytochrome c maturation, or are altogether of unknown function. We also found that signal transduction and redox functions are enriched in the set of Tn5 hits outside the MAI, suggesting that such processes are particularly important in support of magnetosome biosynthesis. IMPORTANCE Magnetospirillum gryphiswaldense is one of the few tractable model magnetotactic bacteria (MTB) for studying magnetosome biomineralization. So far, knowledge on the genetic determinants of this complex process has been mainly gathered using reverse genetics and candidate approaches. In contrast, nontargeted forward genetics studies are lacking, since application of such techniques in MTB has been complicated for a number of technical reasons. Here, we report on the first comprehensive transposon mutagenesis study in MTB, aiming at systematic identification of auxiliary genes necessary to support magnetosome formation in addition to key genes harbored in the magnetosome island (MAI). Our work considerably extends the candidate set of novel subsidiary determinants and shows that the full gene complement underlying magnetosome biosynthesis is larger than assumed. In particular, we were able to define certain cellular pathways as specifically important for magnetosome formation that have not been implicated in this process so far.