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Gene delivery into the plant mitochondria via organelle-specific peptides
Trevor MacMillan,Alicja Ziemienowicz,Fengying Jiang,François Eudes,Igor Kovalchuk 한국식물생명공학회 2019 Plant biotechnology reports Vol.13 No.1
We report a method for genetically engineering the mitochondria of plant cells. Several peptides selected from sorting signal sequences of plant mitochondrial proteins were tested for cell-penetrating, specific organelle-targeting and nucleic acid-binding properties. Selected sequences were named mitochondrial targeting peptides (mTPs). Five of them were used to deliver a linear dsDNA gene construct into AC Ultima spring triticale (X. Triticosecale Wittmack) protoplasts and microspores. The mitochondrial transient expression of the aadA:gfp reporter gene was qualitatively determined with confocal microscopy and quantitatively measured using qRT-PCR. Significant aadA:gfp transcript abundance was reported in protoplasts from deliveries of all five mTPs, while this transcript abundance remained low in microspores. mTP1 transfected microspores were cultured to produce green plantlets and screened for aadA:gfp by endpoint PCR. Targeted gene integration into the mitochondrial genome of eight regenerated haploid triticale lines was confirmed by sequencing. This represents the first report of a successful in vivo transfection of crop plant somatic and germ cells via mitochondrial peptides and a method that can be used to study transient gene expression and to stably deliver genes into the mitochondria of a plant.
( Lili Tian ),( Xinliang Wu ),( Hangqian Yu ),( Fengying Yang ),( Jian Sun ),( Tiezhong Zhou ),( Hong Jiang ) 한국미생물생명공학회 2022 Journal of microbiology and biotechnology Vol.32 No.10
The rise of methicillin-resistant Staphylococcus aureus (MRSA) has resulted in significant morbidity and mortality, and clinical treatment of MRSA infections has become extremely difficult. Sortase A (SrtA), a virulence determinant that anchors numerous virulence-related proteins to the cell wall, is a prime druggable target against S. aureus infection due to its crucial role in the pathogenicity of S. aureus. Here, we demonstrate that isovitexin, an active ingredient derived from a variety of traditional Chinese medicines, can reversibly inhibit SrtA activity in vitro with a low dose (IC<sub>50</sub>=24.72 μg/ml). Fluorescence quenching and molecular simulations proved the interaction between isovitexin and SrtA. Subsequent point mutation experiments further confirmed that the critical amino acid positions for SrtA binding to isovitexin were Ala-92, Ile-182, and Trp-197. In addition, isovitexin treatment dramatically reduced S. aureus invasion of A549 cells. This study shows that treatment with isovitexin could alleviate pathological injury and prolong the life span of mice in an S. aureus pneumonia model. According to our research, isovitexin represents a promising lead molecule for the creation of anti-S. aureus medicines or adjuncts.
( Xiaofeng Zhou ),( Jianghui Li ),( Weilong Wang ),( Fan Yang ),( Bingqian Fan ),( Chenlu Zhang ),( Xiaojun Ren ),( Feng Liang ),( Rong Cheng ),( Fengying Jiang ),( Huaibin Zhou ),( Juanjuan Yang ),( 한국미생물 · 생명공학회 2020 Journal of microbiology and biotechnology Vol.30 No.7
Various genetically engineered microorganisms have been developed for the removal of heavy metal contaminants. Metal biosorption by whole-cell biosorbents can be enhanced by overproduction of metal-binding proteins/peptides in the cytoplasm or on the cell surface. However, few studies have compared the biosorption capacity of whole cells expressing intracellular or surface-displayed metal-adsorbing proteins. In this study, several constructs were prepared for expressing intracellular and surface-displayed Ochrobactrum tritici 5bvl1 ChrB in Escherichia coli BL21(DE3) cells. E. coli cells expressing surface-displayed ChrB removed more Cr(VI) from aqueous solutions than cells with cytoplasmic ChrB under the same conditions. However, intracellular ChrB was less susceptible to variation in extracellular conditions (pH and ionic strength), and more effectively removed Cr(VI) from industrial wastewater than the surface-displayed ChrB at low pH (<3). An adsorptiondesorption experiment demonstrated that compared with intracellular accumulation, cell-surface adsorption is reversible, which allows easy desorption of the adsorbed metal ions and regeneration of the bioadsorbent. In addition, an intrinsic ChrB protein fluorescence assay suggested that pH and salinity may influence the Cr(VI) adsorption capacity of ChrB-expressing E. coli cells by modulating the ChrB protein conformation. Although the characteristics of ChrB may not be universal for all metal-binding proteins, our study provides new insights into different engineering strategies for whole-cell biosorbents for removing heavy metals from industrial effluents.