Sun Chang Kim

Characterization and cDNA cloning of two glycine- and histidine-rich antimicrobial peptides from the roots of shepherd's purse, Capsella bursa-pastoris

Two novel antimicrobial peptides were isolated and characterized from the roots of shepherds purse, Capsella bursa-pastoris. These antimicrobial peptides, named shepherin I and shepherin II, consist of 28 and 38 amino acids, respectively, and are glycine- and histidine-rich peptides. Shepherin I and shepherin II have 67.9% and 65.8% (mol/mol) glycine, respectively, and 28.6% and 21.1% (mol/mol) histidine, respectively. Both shepherins have a Gly-Gly-His motif. These antimicrobial peptides exhibit antimicrobial activity against Gram-negative bacteria and fungi. Circular dichroism spectra of shepherin I and shepherin II showed that shepherin I and shepherin II in 50% trifluoroethanol have 66.7% and 75% random coils, respectively, without any α-helices. cDNA sequence analysis revealed that shepherin I and shepherin II are produced from a single polypeptide, designated shep-GRP, consisting of 120 amino acids; shep-GRP has five distinct domains, an amino-terminal putative signal peptide, a shepherin I, a linker dipeptide, a shepherin II and a carboxy-terminal peptide. Southern blot analysis indicates that the gene encoding shepherins belongs to a low-complexity gene family. Northern blot analysis revealed that transcripts of shep-GRP are present in roots but not in leaves and stems.

Emerging tools for synthetic genome design

Synthetic biology is an emerging discipline for designing and synthesizing predictable, measurable, controllable, and transformable biological systems. These newly designed biological systems have great potential for the development of cheaper drugs, green fuels, biodegradable plastics, and targeted cancer therapies over the coming years. Fortunately, our ability to quickly and accurately engineer biological systems that behave predictably has been dramatically expanded by significant advances in DNA-sequencing, DNA-synthesis, and DNA-editing technologies. Here, we review emerging technologies and methodologies in the field of building designed biological systems, and we discuss their future perspectives.

Synthesis of cross-linked protein-metal hybrid nanoflowers and its application in repeated batch decolorization of synthetic dyes

Herein, we report the preparation of a cross-linked protein-metal hybrid nanoflower (NF) system for laccase immobilization. The immobilized laccase showed effective encapsulation yield and activity recovery of 78.1% and 204%, respectively. The catalytic efficiency (kcatVmax-1) of cross-linked NF (CL-NF) was 2.2-fold more than that of free laccase. The CL-NF also exhibited significantly higher stability towards pH and temperature changes. It exhibited excellent storage stability and tolerance towards solvents and inhibitors as compared with the free enzyme. After 10 cycles of reuses, the NF and CL-NF laccase showed 41.2% and 92.3% residual activity, respectively. The CL-NF showed high oxidation potential, 265% that of the free enzyme, towards phenolic compounds. The CL-NF laccase retained the residual decolorization efficiency of up to 84.6% for synthetic dyes under repeated batch conditions of 10 cycles. These results suggested that the preparation of CL-NF is an effective approach to enhance the enzymatic properties and has great potential in many industrial applications.

Escherichia coli Genome Engineering and Minimization forthe Construction of a Bioengine

A profusion of diverse genome-related information has been obtained by the sequencing of genomes from many microorganisms, functional analyses of these genomes, and the application of bioinformatics techniques to genomics, proteomics, and systems biology. The resulting barrage of data coupled with large-scale gene inactivation studies have allowed researchers to produce a genetic blueprint for a streamline, custom-designed microbe that carries the minimal gene set required for the organism to replicate in a given environment. On the basis of this minimal genome information, several research groups have generated minimal-genome Escherichia coli strains using sophisticated genome engineering techniques, such as the dual transposition, site-specific recombinations, and markerless genome recombination. These minimal genomes display various desirable traits for biological researches, such as improved genome stability, increased transformation efficacy, and higher production of biological materials. Therefore, the generation of a large number of deletion mutants of the minimal E. coli genomes coupled with restructuring of regulatory circuits may lead to facilitate the construction of a variety of custom-designed bacterial strains (also called a “bioengine”) with myriad practical and commercial applications.

Direct protein-protein conjugation by genetically introducing bioorthogonal functional groups into proteins.

Proteins often function as complex structures in conjunction with other proteins. Because these complex structures are essential for sophisticated functions, developing protein-protein conjugates has gained research interest. In this study, site-specific protein-protein conjugation was performed by genetically incorporating an azide-containing amino acid into one protein and a bicyclononyne (BCN)-containing amino acid into the other. Three to four sites in each of the proteins were tested for conjugation efficiency, and three combinations showed excellent conjugation efficiency. The genetic incorporation of unnatural amino acids (UAAs) is technically simple and produces the mutant protein in high yield. In addition, the conjugation reaction can be conducted by simple mixing, and does not require additional reagents or linker molecules. Therefore, this method may prove very useful for generating protein-protein conjugates and protein complexes of biochemical significance.

Development of Bacillus methanolicus methanol dehydrogenase with improved formaldehyde reduction activity

Methanol dehydrogenase (MDH), an NAD+-dependent oxidoreductase, reversibly converts formaldehyde to methanol. This activity is a key step for both toxic formaldehyde elimination and methanol production in bacterial methylotrophy. We mutated decameric Bacillus methanolicus MDH by directed evolution and screened mutants for increased formaldehyde reduction activity in Escherichia coli. The mutant with the highest formaldehyde reduction activity had three amino acid substitutions: F213V, F289L, and F356S. To identify the individual contributions of these residues to the increased reduction activity, the activities of mutant variants were evaluated. F213V/F289L and F213V/F289L/F356S showed 25.3- and 52.8-fold higher catalytic efficiency (kcat/Km) than wild type MDH, respectively. In addition, they converted 5.9- and 6.4-fold more formaldehyde to methanol in vitro than the wild type enzyme. Computational modelling revealed that the three substituted residues were located at MDH oligomerization interfaces, and may influence oligomerization stability: F213V aids in dimer formation, and F289L and F356S in decamer formation. The substitutions may stabilise oligomerization, thereby increasing the formaldehyde reduction activity of MDH.

Rerouting of NADPH synthetic pathways for increased protopanaxadiol production in Saccharomyces cerevisiae

Ginseng (Panax ginseng) and its bioactive components, ginsenosides, are popular medicinal herbal products, exhibiting various pharmacological effects. Despite their advocated use for medication, the long cultivation periods of ginseng roots and their low ginsenoside content prevent mass production of this compound. Yeast Saccharomyces cerevisiae was engineered for production of protopanaxadiol (PPD), a type of aglycone characterizing ginsenoside. PPD-producing yeast cell factory was further engineered by obtaining a balance between enzyme expressions and altering cofactor availability. Different combinations of promoters (PGPD, PCCW12, and PADH2) were utilized to construct the PPD biosynthetic pathway. Rerouting the redox metabolism to improve NADPH availability in the engineered S. cerevisiae also increased PPD production. Combining these approaches resulted in more than an 11-fold increase in PPD titer over the initially constructed strain. The series of metabolic engineering strategies of this study provides a feasible approach for the microbial production of PPD and development of microbial platforms producing other industrially-relevant terpenoids.

VEGF siRNA Delivery by a Cancer-Specific Cell-Penetrating Peptide

RNA interference provides an effective tool for developing antitumor therapies. Cell-penetrating peptides (CPPs) are delivery vectors widely used to efficiently transport small-interfering RNA (siRNA) to intracellular targets. In this study, we investigated the efficacy of the cancer-specific CPP carrier BR2 to specifically transport siRNA to cancer-target cells. Our results showed that BR2 formed a complex with anti-vascular endothelial growth factor siRNA (siVEGF) that exhibited the appropriate size and surface charge for in vivo treatment. Additionally, the BR2-VEGF siRNA complex exhibited significant serum stability and high levels of gene-silencing effects in vitro. Moreover, the transfection efficiency of the complex into a cancer cell line was higher than that observed in non-cancer cell lines, resulting in downregulated intracellular VEGF levels in HeLa cells and comprehensively improved antitumor efficacy in the absence of significant toxicity. These results indicated that BR2 has significant potential for the safe, efficient, and specific delivery of siRNA for diverse applications.