Surface Display of Complex Enzymes by <i>in Situ</i> SpyCatcher‐SpyTag Interaction

Gallus, Sabrina; Peschke, Theo; Paulsen, Malte; Burgahn, Teresa; Niemeyer, Christof M.; Rabe, Kersten S.

doi:10.1002/cbic.202000102

Cited by 30 publications

(42 citation statements)

References 44 publications

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“…OMVs have proven their value in the development of a vaccine against Neisseria meningitis and are increasingly investigated as a vaccine against other pathogens [15]. Furthermore, the use of engineered OMVs has emerged as vehicles for targeted drug delivery [11]. In this article, it was shown that OMVs can serve as an in vivo immobilization strategy for recombinant production of self-sufficient CYP monooxygenases, thereby proposing an alternative production strategy.…”

Section: Discussionmentioning

confidence: 99%

“…Thirdly, a recent article reports the display of CYP102A1 by in situ SpyCatcher-SpyTag interaction. [11].…”

Section: Introductionmentioning

confidence: 99%

“…OMVs are formed by blebbing of the OM. This blebbing is thought to occur at regions where peptidoglycan:membrane crosslinks are reduced, leading to decreased envelope stability [11], [18], [19]. Non-covalent interactions between the peptidoglycan (PG) layer and the Tol-Pal complex serves as one of the crosslinks, providing envelope stability.…”

Section: Introductionmentioning

confidence: 99%

“…Proper crosslinking was therefore prevented, consequently leading to hypervesiculation [21], [22]. Furthermore, OMV biogenesis occurs as a response to external factors such as temperature, nutrient availability and quorum sensing [11], [18], [19]. Also, several antibiotics have been shown to induce OMV production.…”

Section: Introductionmentioning

confidence: 99%

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Display of the self-sufficient CYP102A1 on the surface of E. coli-derived Outer Membrane Vesicles

Devriese

Surmont

Lynen

et al. 2021

Preprint

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The cytochrome P450 (CYP) monooxygenase superfamily offers the unique ability to catalyze regio-and stereospecifical oxidation of a non-activated C-H bond. CYPs found applications in the synthesis of pharmaceuticals and drug metabolites as well as in bioremediation. They are typically used in whole-cell bioconversion processes, due to their low stability and the need for a redox partner and cofactor. Unfortunately, substrate uptake and/or product transport limitations are frequently encountered and side reactions occur due to other enzymes in the cellular environment. Here, we present a proof-of-principle of a novel cell-free cytochrome P-450 nanocatalyst based on surface display on bacterial outer membrane vesicles. The self-sufficient CYP 102A1 from Bacillus megaterium was engineered to be translocated on the outer membrane vesicles of Escherichia coli. The resulting vesicles can simply be isolated from the culture supernatant. Moreover, no expensive and elaborate enzyme purification is required. This approach shows great promise as an alternative strategy to recombinantly produce CYP enzymes for a variety of applications, such as in fine chemical production and in the development of biosensors.

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Section: Discussionmentioning

confidence: 99%

“…Thirdly, a recent article reports the display of CYP102A1 by in situ SpyCatcher-SpyTag interaction. [11].…”

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Display of the self-sufficient CYP102A1 on the surface of E. coli-derived Outer Membrane Vesicles

Devriese

Surmont

Lynen

et al. 2021

Preprint

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“…The SpyTag/SpyCatcher system has been used to immobilize foreign proteins onto nanoparticles 25 – 27 and bacterial outer membrane vesicles 25 – 30 . Recently, cell surface display of enzymes in E. coli using SpyTag/SpyCatcher has been reported 31 , but there was no example of displaying fusion proteins produced by post-translational ligation in eukaryotic cells.…”

Section: Introductionmentioning

confidence: 99%

Development of a yeast cell surface display method using the SpyTag/SpyCatcher system

Kajiwara

Aoki

Koike³

2021

Sci Rep

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Yeast cell surface display (YSD) has been used to engineer various proteins, including antibodies. Directed evolution, which subjects a gene to iterative rounds of mutagenesis, selection and amplification, is useful for protein engineering. In vivo continuous mutagenesis, which continuously diversifies target genes in the host cell, is a promising tool for accelerating directed evolution. However, combining in vivo continuous evolution and YSD is difficult because mutations in the gene encoding the anchor proteins may inhibit the display of target proteins on the cell surface. In this study, we have developed a modified YSD method that utilises SpyTag/SpyCatcher-based in vivo protein ligation. A nanobody fused with a SpyTag of 16 amino acids and an anchor protein fused with a SpyCatcher of 113 amino acids are encoded by separate gene cassettes and then assembled via isopeptide bond formation. This system achieved a high display efficiency of more than 90%, no intercellular protein ligation events, and the enrichment of target cells by cell sorting. These results suggested that our system demonstrates comparable performance with conventional YSD methods; therefore, it can be an appropriate platform to be integrated with in vivo continuous evolution.

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Artificial Spores as Multi‐Functional Biocatalysts to Perform Biosynthetic Cascades

Iturralde,

Ripoll,

Silvio

et al. 2024

Adv Funct Materials

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Cells exhibit diverse structural formations such as biofilms and spores, enabling them to acquire novel functionalities. Many of these structures display biomacromolecules, including enzymes, tethered to cell walls to support various extracellular processes. Alternatively, encapsulating single cells with polymer coatings offers a strategy that circumvents the need for genetic engineering while imparting artificial functionalities to cells. Here, a universal method is presented for encapsulating single gram‐negative microbes with polymeric coatings based on the ancestral gall ink formed by tannic acid‐iron complexes. As a result, synthetic spores are achieved that selectively bind His‐tagged enzymes through the formation of unprecedented galloyl/imidazole‐Fe2+ complexes via ligand substitution demonstrated by density functional theory. These synthetic spores with a thickness of 41.5 ± 4.2 nm and a stiffness of 6.0 ± 3.5 GPa serve as biocatalytic materials for the one‐pot oxidative amination of diols into amino alcohols, facilitated by the cooperative catalysis between intracellular endogenous or recombinant oxidoreductases, and an extracellular transaminase from Pseudomonas fluorescens displayed at the spore surface. These spores maintain their performance in three consecutive batch cycles. Integrating isolated enzymes onto the surface of engineered microbes coated with polymers offers novel opportunities for synthetic biology, advancing the efficiency of biosynthetic cascades in solid‐state environments.

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Surface Display of Complex Enzymes by in Situ SpyCatcher‐SpyTag Interaction

Cited by 30 publications

References 44 publications

Display of the self-sufficient CYP102A1 on the surface of E. coli-derived Outer Membrane Vesicles

Display of the self-sufficient CYP102A1 on the surface of E. coli-derived Outer Membrane Vesicles

Development of a yeast cell surface display method using the SpyTag/SpyCatcher system

Artificial Spores as Multi‐Functional Biocatalysts to Perform Biosynthetic Cascades

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