Generation of multifunctional magnetic nanoparticles with amplified catalytic activities by genetic expression of enzyme arrays on bacterial magnetosomes

TLDR: In this paper, a new in vivo strategy is explored for magnetosome display of foreign polypeptides with maximized protein-to-particle ratios, where 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.

Abstract: 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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Table 2. Particle sizes of magnetosomes isolated from mamC-(gusA)1-5-megfp mutant strains. Diameters were determined by TEM (n = 200) and zetasizer analysis (three independent measurements). Particle suspensions were analyzed in 10 mM Hepes / 1 mM EDTA at pH 7.2.

Table 3. Protein amounts, copy numbers and enzymatic activities of magnetosomes displaying GusA arrays on the particle surface. Denaturing PAGE with subsequent quantitative Western blotting was used to estimate the molecular masses of the fusions according to their electrophoretic mobility. The obtained values are compared with the theoretical masses based on amino acid composition. Furthermore, magnetosomes were subjected to activity assays to determine KM and vmax (3.3 µg Fe) (Figure S2; SI). GusA protein amounts (calculated by densitometrical analysis of PVDF membranes, Figure 5A) were subsequently used to calculate specific enzymatic activities.

Figure 1. (A) Schematic representation showing the genetic organization of the constructed expression cassettes for the display of MamC-(GusA)n-mEGFP fusions on the magnetosome surface. (B) The resulting particles displayed arrays of one to five copies of GusA and mEGFP as fluorophore and additional translational reporter. MamC, GusA monomers and mEGFP were fused via G10 linkers composed of ten glycine residues. (Size of particles and proteins not to scale)

Figure 2. Magnetosome expression of MamC-GusA or MamC-GusA-mEGFP fusions. (A) Transmission electron micrographs of WT::mamC-gusA-megfp and ∆mamC::mamC-gusA-megfp cells indicate a wild type - like, chain - like arrangement of magnetosomes, positioned at midcell. mEGFP expression was confirmed by fluorescence microscopy (insets), showing fluorescence at magnetosome chain position. (B) Glucuronide-hydrolyzing activity of MamC-GusA-mEGFP magnetosomes. In the assay, p-nitrophenol-β-D-glucuronide was cleaved by GusA, yielding 3- glucuronate and p-nitrophenol. Formation of the latter was monitored by measuring the absorption increase at 415 nm and resulted in a characteristic yellow color of the supernatant (insets). Enzymatic activities were normalized and are given as A/A0 ratios, with A0 representing the initial activity. (C) Bar chart illustrating cellular mEGFP fluorescence, particle-displayed GusA amounts and GusA reaction rates (vmax) of the indicated M. gryphiswaldense strains. Fluorescence was normalized to cell density and reported as relative fluorescence units (RFU). For quantification of GusA on the particle surface, isolated magnetosomes were subjected to denaturing PAGE (1.5 or 3 µg Fe / lane) followed by quantitative Western blotting employing an IgG antibody directed against GusA. GusA reaction rates (vmax) were calculated as the mean of Michaelis-Menten, LineweaverBurk and Hanes-Woolf approximations. Error bars represent standard deviations calculated from at least three independent measurements.

Figure 4. Upper panel: TEM of representative cells of the wild type and the mutant strains mamC(gusA)1-5-megfp of M. gryphiswaldense. Wild type - like particle arrangement of one or two magnetosome chains positioned at midcell were observed in all strains. Lower panel: TEM of isolated, negatively stained magnetosomes revealed an organic layer of up to 10.9 ± 4.0 nm in thickness, thereby increasing the particle diameter and the distances between magnetite cores. Arrows indicate junctions of up to 30 nm between particles of mamC-(gusA)2-5-megfp fusions.

Figure 3. Enzymatic activity of isolated MamC-GusA-mEGFP magnetosomes after several cycles of centrifugation and resuspension. The time-dependent production of p-nitrophenol through hydrolysis of the artificial substrate p-nitrophenyl-β-D-glucuronide was monitored, and absorption values were normalized for direct comparison. Insets: Magnetosome pellets exhibiting glucuronidehydrolyzing activity accumulated next to the pole of an external permanent magnet.

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Mickoleit, F., & Schüler, D. (2018). Generation of multifunctional magnetic nanoparticles
with amplified catalytic activities by genetic expression of enzyme arrays on bacterial
magnetosomes. Advanced Biosystems, 2(1), 1700109
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Generation of Multifunctional Magnetic Nanoparticles with Amplified Catalytic Activities by
Genetic Expression of Enzyme Arrays on Bacterial Magnetosomes
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Frank Mickoleit, and Dirk Schüler*
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Abstract
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:8 
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1. Introduction
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Magnetospirillum gryphiswaldense  A)
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3
C 
1 A2!F+C
 !!
!!
D!2E

(  
DFE
 ;
   9
9+*B,
DGE
%
  


DE
 
DE
!
 
D1E
 
D32E

A*H?C
DF!E
A*,?C
DE

> ;
#9in vitro
D!3E
+
 ;. 
 in vivo)
8A*C*#
AI*1C&*# !
9
D2FE

DGE
 
A2!+(C8 B!
 
D2E
 
 
DG1E


 
1

,*#8
A4),C
D11E
 AC

D1E

)!

DE
*#8 4!

D11E
*>#?8
D13E
9 )
8*#M. magneticum
 *F*& 

D11E
)89 !
HB,9  
 8
D12E
8:
D1FE

>8  9  
8?A
!C9  
  89!!
 ,8 
).!+
DGE

8 9,
(#

AH.%CmamC?!9
4),8AI54),CG!8
>8  8 
*# ?
 .
DGE
4),
*#
84),

 
3

? 8  8
9 4&AJ!Escherichia coliC
M. gryphiswaldense  

DG11GE
"  4&
54), *#
,854),
G! 4&8! 
) ; 
4& 
  !8
   

2. Results
2.1. Magnetosomal expression of GusA-mEGFP fusions results in magnetic fluorescent
particles with stable catalytic activity
4&4&!54),*#mamC-(gusA)
n
-megfp
9,
(#32
9
AH.%C
DGE
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KmamCmamC-gusA-megfpmamC-gusA-megfp 2
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Frequently asked questions

Q1. What are the contributions in "Generation of multifunctional magnetic nanoparticles with amplified catalytic activities by genetic expression of enzyme arrays on bacterial magnetosomes" ?

Assuming layers of GusA rows surrounding the particles, the monomers would thus cover up to 90 % of the magnetosome surface. Their approach generates nanoparticles that exhibit magnetism, fluorescence and stable catalytic activities, which were step-wise 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. 

Q2. What is the role of GusA in the synthesis of biohybrid nano?

GusA is a cofactor-independent acid hydrolase that catalyzes the cleavage of 3-glucuronides, yielding 3-glucuronates and an alcohol. 

Q3. What is the role of the GusA enzyme in the fusion of spores?

Since the GusA enzyme is likely to function as a 272-kDa homotetramer,[41,45,46] the catalytic activity of these fusions depends on the proper folding of the fused GusA monomers and their assembly into GusA tetramers. 

Q4. What other proteins were exploited for the magnetosome display?

MamC was also exploited for the magnetosomal display of immunoglobulin Gbinding domains,[33] single chain MHCI/peptide complexes[34] and enzyme proteins. 

Q5. What is the effect of the fusion of GusA monomers on the spore?

Due to an increased proximity of single GusA monomers, tetramer formation might be even facilitated, as suggested by decreased KM values formagnetosomal GusA compared to soluble expressed monomers. 

Q6. How did the fusion of GusA monomers in M. gryphiswald?

In their study, chromosomal insertion of fusions with multiple copies of GusA in M. gryphiswaldense led to up to 18% (w/w) enzyme loading of the magnetosomes and a nearly 3-fold increased catalytic activity (relative to single copy expression) using genetic multiplication of GusA monomers fused as large hybrid proteins to abundant MamC anchors. 

Q7. What are the challenges of displaying multimeric enzyme proteins on magnetosome particles?

Since the stabile expression of catalytically active enzyme proteins, their immobilization and re-usability arestill common challenges,[50] this provides a highly promising route also for the display of other enzyme proteins and peptides more relevant for both biotechnological, biomedical and research applications. 

Q8. How did Borg and co-workers achieve the increased expression of mamC?

In combination with codon-optimization of the reporter eGFP for magnetobacterial expression (= mEGFP) this led to 2.8-fold increased expression levels. 

Q9. What is the role of eGFP in the magnetosome?

In addition, potential application (e.g. as magnetic sensors or as bi-/multimodal contrast agents) and functionalization of magnetosomes rely on densely decorated, catalytically active particles with maximized protein-to-particle ratios, which has been difficult to achieve by genetic means. 

Q10. How can multimeric enzymes be displayed on magnetosome particles?

In this study, the authors have demonstrated that multimeric enzymes can be displayed on magnetosome particles in high copy numbers, thereby multiplying their coverage by 5 and nearly triplicating their specific activity per particle. 

Q11. How did they find the activity of GusA?

Sheppard et al. (2011) observed a high activity for GusA when immobilized to living diatom silica by genetic fusion to silaffin protein, resulting in about 0.1% (w/w) enzyme loading of the silica.[44] 

Q12. What is the enzymatic stability of GusA?

The enzymatic stability of functionalized MamC-GusA-mEGFP magnetosomes was investigated by subjecting them to multiple cycles of collection and re-addition of fresh substrate. 

Q13. What was the effect of TEM analysis on the particle diameters of isolated magnetosomes?

TEM analysis also showed an increased spacing between purified magnetosome particles, and electron-light junctions of up to 30 nm were occasionally observed between isolated MamC-(GusA)2-5-eGFP magnetosomes (Figure 4). 

Q14. What are the phospholipids in a magnetosome?

This so-called magnetosome membrane consists of phospholipids and a set of magnetosome-specific, membrane-associated proteins.[1-5]