Cell-penetrating peptides (CPPs) have proven very effective as intracellular delivery vehicles for various therapeutics. However, there are some concerns about non-specific penetration and cytotoxicity of CPPs for effective cancer treatments. Herein, based on the cell-penetrating motif of an anticancer peptide, buforin IIb, we designed several CPP derivatives with cancer cell specificity. Among the derivatives, a 17-amino acid peptide (BR2) was found to have cancer-specificity without toxicity to normal cells. After specifically targeting cancer cells through interaction with gangliosides, BR2 entered cells via lipid-mediated macropinocytosis. Moreover, BR2 showed higher membrane translocation efficiency than the well-known CPP Tat (49–57). The capability of BR2 as a cancer-specific drug carrier was demonstrated by fusion of BR2 to a single-chain variable fragment (scFv) directed toward a mutated K-ras (G12V). BR2-fused scFv induced a higher degree of apoptosis than Tat-fused scFv in K-ras mutated HCT116 cells. These results suggest that the novel cell-penetrating peptide BR2 has great potential as a useful drug delivery carrier with cancer cell specificity.

The genomic stability and integrity of host strains are critical for the production of recombinant proteins in biotechnology. Bacterial genomes contain numerous jumping genetic elements, the insertion sequences (ISs) that cause a variety of genetic rearrangements, resulting in adverse effects such as genome and recombinant plasmid instability. To minimize the harmful effects of ISs on the expression of recombinant proteins in Escherichia coli, we developed an IS-free, minimized E. coli strain (MS56) in which about 23 % of the genome, including all ISs and many unnecessary genes, was removed. Here, we compared the expression profiles of recombinant proteins such as tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and bone morphogenetic protein-2 (BMP2) in MG1655 and MS56. Hopping of ISs (IS1, IS3, or IS5) into the TRAIL and BMP2 genes occurred at the rate of ~10(-8)/gene/h in MG1655 whereas such events were not observed in MS56. Even though IS hopping occurred very rarely (10(-8)/gene/h), cells containing the IS-inserted TRAIL and BMP2 plasmids became dominant (~52 % of the total population) 28 h after fermentation began due to their growth advantage over cells containing intact plasmids, significantly reducing recombinant protein production in batch fermentation. Our findings clearly indicate that IS hopping is detrimental to the industrial production of recombinant proteins, emphasizing the importance of the development of IS-free host strains.

Escherichia coli has been explored as a host for butanol production because of its many advantages such as a fast growth and easy genetic manipulation. Butanol toxicity, however, is a major concern in the biobutanol production with E. coli. In particular, E. coli growth is severely inhibited by butanol, being almost completely stopped by 1% (vol/vol) butanol. Here we developed a new method to increase the butanol-tolerance of E. coli with artificial transcription factor (ATF) libraries which consist of zinc finger (ZF) DNA-binding proteins and an E. coli cyclic AMP receptor protein (CRP). Using these ATFs, we selected a butanol-tolerant E. coli which can tolerate up to 1.5% (vol/vol) butanol, with a concomitant increase in heat resistance. We also identified genes of E. coli that are associated with the butanol-tolerance. These results show that E. coli can be engineered as a promising host for high-yield butanol production.

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Lipases have been utilized industrially to produce biodiesel, oleochemicals, and pharmaceuticals. Many efforts such as metagenomics, directed evolution and enzyme immobilization have been devoted to enhance the lipase activity.Here, we designed a recombinant lipase, NKC-M37-MAT, that was generated by incorporating an N-terminal amphipathic peptide (NKC) and a C-terminal coiled-coil peptide (MAT) into Photobacterium lipolyticum M37 lipase. The hydrophobic face of NKC improve the accessibility (K m ), and catalytic efficiency (K cat /K m ) of the soluble lipase toward hydrophobic substrate and tetrameric MAT further enhanced lipase catalytic activity (U/mg) through cooperative binding to its substrate such that the catalytic activity (U/mg) of NKC-M37-MAT was increased by a maximum of 54-fold compared with the wildtype, which decreased the biodiesel production time 5-fold from 30 h to 6 h. This novel approach shows promise as a platform technology to increase lipase catalytic efficiency for industrial-scale production of biodiesel and biochemicals synthesized from hydrophobic substrates.Triacylglycerol lipase (E.C. 3.1.1.3), 1 which catalyzes the hydrolysis of triglycerides to fatty acids and glycerols, plays an important role in industries that produce products such as detergents, food, leather, paper, cosmetics, and bioenergy. 1-3 Because of their broad substrate specificity and enantioselectivity, lipases have been utilized by the pharmaceutical industry to synthesize pure R-and S-optical isomers. 4 The energy industry is also using lipases to produce biodiesel from vegetable oil as well. 1,3,5,6 Biodiesel products, an emerging renewable energy source, are produced through trans-esterification in the presence of a catalyst which could be basic (generally, NaOH), acid (usually, HCl or H 2 SO 4 ), or enzymatic. 7-9 However, base-catalyzed trans-esterification is not feasible since alkaline can react with free fatty acid to form unwanted soap and water, which would affect the biodiesel quality and require extra investment for downstream separation and purification. In acid-catalyzed esterification, large excess amount of alcohol is required in order to reach high biodiesel yield, and if sulfuric acid is utilized as catalysts, it is difficult to remove them after reaction. Moreover, chemical-catalyzed trans-esterification produces toxic wastewater and consumes high amounts of energy to maintain high reaction temperature. 8 To overcome these problems, enzymatic methods using lipase are receiving special attention as an attractive alternative approach for biodiesel production. The great advantages of lipases compared with chemical catalysts are as follows: 1) relatively low reaction temperatures (approximately 40°C), 6 2) fewer process steps that generate reduced amounts of hazardous materials, 3) improved product separation that generates higher purity glycerol. 5,6 Since the enzymatic synthesis of biodiesel occurs at the lipid-water interface, increasing the accessibility of water-soluble lipase such as M37 to hydro...

Cell-penetrating peptides (CPPs) have proven very effective as intracellular delivery vehicles for various therapeutics. However, there are some concerns about non-specific penetration and cytotoxicity of CPPs for effective cancer treatments. Herein, based on the cell-penetrating motif of an anticancer peptide, buforin IIb, we designed several CPP derivatives with cancer cell specificity. Among the derivatives, a 17-amino acid peptide (BR2) was found to have cancer-specificity without toxicity to normal cells. After specifically targeting cancer cells through interaction with gangliosides, BR2 entered cells via lipid-mediated macropinocytosis. Moreover, BR2 showed higher membrane translocation efficiency than the well-known CPP Tat (49-57). The capability of BR2 as a cancer-specific drug carrier was demonstrated by fusion of BR2 to a single-chain variable fragment (scFv) directed toward a mutated K-ras (G12V). BR2-fused scFv induced a higher degree of apoptosis than Tat-fused scFv in K-ras mutated HCT116 cells. These results suggest that the novel cell-penetrating peptide BR2 has great potential as a useful drug delivery carrier with cancer cell specificity.

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