Biomanufacturing Biotinylated Magnetic Nanomaterial via Construction and Fermentation of Genetically Engineered Magnetotactic Bacteria

Abstract

Biosynthesis provides a critical way to deal with global sustainability issues and has recently drawn increased attention. However, modifying biosynthesized magnetic nanoparticles by extraction is challenging, limiting its applications. Magnetotactic bacteria (MTB) synthesize single-domain magnetite nanocrystals in their organelles, magnetosomes (BMPs), which are excellent biomaterials that can be biologically modified by genetic engineering. Therefore, this study successfully constructed in vivo biotinylated BMPs in the MTB Magnetospirillum gryphiswaldense by fusing biotin carboxyl carrier protein (BCCP) with membrane protein MamF of BMPs. The engineered strain (MSR-∆F-BF) grew well and synthesized small-sized (20 ± 4.5 nm) BMPs and were cultured in a 42 L fermenter; the yield (dry weight) of cells and BMPs reached 8.14 g/L and 134.44 mg/L, respectively, approximately three-fold more than previously reported engineered strains and BMPs. The genetically engineered BMPs (BMP-∆F-BF) were successfully linked with streptavidin or streptavidin-labelled horseradish peroxidase and displayed better storage stability compared with chemically constructed biotinylated BMPs. This study systematically demonstrated the biosynthesis of engineered magnetic nanoparticles, including its construction, characterization, and production and detection based on MTB. Our findings provide insights into biomanufacturing multiple functional magnetic nanomaterials.

Keywords: biomaterials; biosynthesis; engineered magnetosome; fermentation; magnetotactic bacteria.

Figure 1

Construction and growth of the engineered bacteria MSR−∆F−BF and BMP synthesis detection. (A) Schematic diagram of MSR−∆F−BF construction and biotinylated BMP extraction. (a) construction of bccp and mamF genes fusion expression vector (pBBR−bccp−mamF); (b) transformation of pBBR−bccp−mamF to MSR−∆F by biparental conjugation to construct the engineered strain (MSR−∆F−BF); (c) in vivo biotinylated BMP by biotin ligase linkage of biotin to BCCP on BMP; (d) ultrasonic homogenizer-disrupted cell; (e) magnetically separated biotinylated BMP from cell debris. (B) Western blotting detects the fusion protein display on engineered BMP. (C) TEM micrograph of MSR−∆F, MSR−∆F−BF, and MSR−WT; there were no BMPs in MSR−∆F cell, small−sized BMPs in MSR−∆F−BF cell, and normal particle size BMPs in MSR−WT cell. The results indicated that the fusion gene was successfully expressed and complemented the disruption of the mamF gene in MSR−∆F−BF, suggesting MSR−∆F−BF could synthesize of BMPs successfully. (D) Cell growth curve (D1) and Cmag curve (D2); MSR−∆F−BF cell growth rate was close to MSR−WT and faster than MSR−∆F. Cmag value was lower in MSR−WT.

Figure 2

(A) High−resolution TEM and fast Fourier transform analysis of the crystal composition of BMP−∆F−BF; the core constituent of BMP−∆F−BF was Fe₃O₄ NPs. (B) Magnetic property analysis of MSR−∆F−BF, S*: static moment.

Figure 3

Submerged culture of MSR−∆F−BF in a 42 L fermenter (A) rpm and dO₂% values as a function of time. As the cell grew, the dO₂% in the broth was consumed continuously. dO₂% decreased to ~5%, and IPTG was added to introduce fusion gene expression. dO₂% decreased to ~1%; the agitation speed was added at 10 rpm/2 h to maintain microaerobic condition. (B) MSR−∆F−BF cell growth and Cmag value curve. The highest OD₅₆₅ and Cmag reached ~22 and ~1.3, respectively.

Figure 4

Purification of BMP–∆F–BF. (A) Magnetic–rack–separated BMP–∆F–BF (arrow). (B) Protein concentration in the supernatant following various numbers of wash cycles. After washing six times, the protein concentration was lower than 0.1 mg/mL, and as the wash cycle increased, the protein concentration stopped decreasing. (C) TEM micrograph of purified BMP–∆F–BF after washing six times. There was no stain on the photo background, and BMP–∆F–BF was well purified. (D) Particle size distribution of BMP–∆F–BF and BMP–WT.

Figure 5

Comparison of streptavidin linkage rate with BMP−∆F−BF, BMP−biotin, and MB−biotin. (A) Optimization of conditions for conjugation of streptavidin to BMP−∆F−BF: (A1) linkage temperature, (A2) linkage buffer, 1:10 mmol/L Tris-HCl, pH 7.4, 2:10 mmol/L Hepes, pH 7.4, 3:10 mmol/L Gly, pH 7.4, 4:10 mmol/L Na₂HPO₃-C₆H₈O₇, pH 7.4, 5:10 mmol/L PBS, pH 7.4; (A3) streptavidin weight. Linkage temperature and buffer have no significant influence on the streptavidin linkage rate of BMP−∆F−BF. When BMP−∆F−BF and streptavidin weight rate was 1:3, BMP−∆F−BF linkage with streptavidin rate was markedly higher than BMP−WT nonspecific absorption (A3), which were suitable conditions for BMP−∆F−BF linkage with streptavidin. (B) Schematic of chemically conjugated biotin to BMP and BMP co-incubated with NHS-biotin. The -NHS group can react with -NH₂ group of BMP modified streptavidin to the surface of BMP, forming biotin-labeled BMP. (C) Comparison of Streptavidin−HRP linkage rate of BMP−∆F−BF, BMP−biotin, and MB−biotin. (D) Comparison of Streptavidin−HRP linkage rate of BMP−∆F−BF, BMP−biotin, and MB−biotin at different storage times. The biosynthesized BMP−∆F−BF linkage rate was more stable than that of BMP−biotin and MB−biotin. Data are presented as the mean  ±  s.d. (n  =  3 biological replicates per group) and statistically analyzed using the two-sided Student’s t-test: ** p < 0.01, *** p < 0.001.