Kouichi Kuroda

Next generation of antimicrobial peptides as molecular targeted medicines.

Antibiotics have significantly improved our living environments. However, overuse of antibiotics has led to the emergence of multi-drug resistant microorganisms, and the subsequent constant demand for the exploration of novel antibiotics. To this end, antimicrobial peptides (AMPs) have attracted much attention as a novel class of antibiotics. AMPs have strong antimicrobial activity against a wide-range of species, including gram-positive and gram-negative bacteria, fungi, and viruses. In addition, they are also effective against pathogenic organisms that are resistant to conventional drugs. Despite their great potential, the hemolytic activity and a highly broad spectrum of activity of AMPs dictate the need for amendments to develop safe pharmaceuticals. The human body contains commensal microflora as an integral part of complex mucosal surfaces that offers protection against pathogenic organisms. Administration of antibiotics with broad spectra of activity disrupts the indigenous microflora and increases the risks of diarrhea and other fatal infections. Therefore, it is difficult, but vital, to develop treatments capable of rapidly eliminating pathogenic organisms while maintaining the commensal microbiota. As such, novel pharmaceuticals, safe designer AMPs have been heavily researched. In this article, we review recent attempts to spatially and temporally regulate AMPs to enhance the quality-of-life of patients.

Effect of pretreatment of hydrothermally processed rice straw with laccase-displaying yeast on ethanol fermentation

A gene encoding laccase I was identified and cloned from the white-rot fungus Trametes sp. Ha1. Laccase I contained 10 introns and an original secretion signal sequence. After laccase I without introns was prepared by overlapping polymerase chain reaction, it was inserted into expression vector pULD1 for yeast cell surface display. The oxidation activity of a laccase-I-displaying yeast as a whole-cell biocatalyst was examined with 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS), and the constructed yeast showed a high oxidation activity. After the pretreatment of hydrothermally processed rice straw (HPRS) with laccase-I-displaying yeast with ABTS, fermentation was conducted with yeast codisplaying endoglucanase, cellobiohydrolase, and β-glucosidase with HPRS. Fermentation of HPRS treated with laccase-I-displaying yeast was performed with 1.21-fold higher activities than those of HPRS treated with control yeast. The results indicated that pretreatment with laccase-I-displaying yeast with ABTS was effective for direct fermentation of cellulosic materials by yeast codisplaying endoglucanase, cellobiohydrolase, and β-glucosidase.

ABC transporters and cell wall proteins involved in organic solvent tolerance in Saccharomyces cerevisiae

Pleiotropic drug resistance 1 (Pdr1p) protein is a key transcription factor in multidrug resistance. Specifically, R821S point mutation in the PDR1 gene in Saccharomyces cerevisiae diploid KK-211 strain plays an important role in tolerance to hydrophobic organic solvents, though the molecular mechanisms underlying organic solvent tolerance are unclear. In KK-211, several ABC transporters and cell wall proteins were upregulated. PDR1 R821S mutant derived from the laboratory haploid strain MT8-1 confirmatively tolerated hydrophobic organic solvents, and this study is the first to reveal its tolerance of the hydrophilic organic solvent, dimethyl sulfoxide (DMSO). To identify the genes involved in the organic solvent tolerance, we focused on the upregulated 4 ABC transporters and 6 cell wall proteins in KK-211, and demonstrated 2 ABC transporters, Pdr10p and Snq2p, and the 4 cell wall proteins, Wsc3p, Pry3p, Pir1p, and Ynl190wp were responsible for hydrophobic organic solvent (n-decane and n-undecane) tolerance. Tolerance to the hydrophilic organic solvent, DMSO, was facilitated by the overproduction of 2 ABC transporters, Snq2p and Yor1p and, 3 cell wall proteins, Wsc3p, Pry3p, and Ynl190wp. Our results suggest that overexpression of genes encoding proteins effective for tolerance to specific organic solvents would enable enhanced tolerances for practical use.

Synthesis of functional dipeptide carnosine from nonprotected amino acids using carnosinase-displaying yeast cells.

Carnosine (β-alanyl-l-histidine) is one of the bioactive dipeptides and has antioxidant, antiglycation, and cytoplasmic buffering properties. In this study, to synthesize carnosine from nonprotected amino acids as substrates, we cloned the carnosinase (CN1) gene and constructed a whole-cell biocatalyst displaying CN1 on the yeast cell surface with α-agglutinin as the anchor protein. The display of CN1 was confirmed by immunofluorescent labeling, and CN1-displaying yeast cells showed hydrolytic activity for carnosine. When carnosine was synthesized by the reverse reaction of CN1, organic solvents were added to the reaction mixture to reduce the water content. The CN1-displaying yeast cells were lyophilized and examined for organic solvent tolerance. Results showed that the CN1-displaying yeast cells retained their original hydrolytic activity in hydrophobic organic solvents. In the hydrophobic organic solvents and hydrophobic ionic liquids, the CN1-displaying yeast cells catalyzed carnosine synthesis, and carnosine was synthesized from nonprotected amino acids in only one step. The results of this research suggest that the whole-cell biocatalyst displaying CN1 on the yeast cell surface can be used to synthesize carnosine with ease and convenience.

Specific adsorption of tungstate by cell surface display of the newly designed ModE mutant

By cell surface display of ModE protein that is a transcriptional regulator of operons involved in the molybdenum metabolism in Escherichia coli, we have constructed a molybdate-binding yeast (Nishitani et al., Appl Microbiol Biotechnol 86:641–648, 2010). In this study, the binding specificity of the molybdate-binding domain of the ModE protein displayed on yeast cell surface was improved by substituting the amino acids involved in oxyanion binding with other amino acids. Although the displayed S126T, R128E, and T163S mutant proteins adsorbed neither molybdate nor tungstate, the displayed ModE mutant protein (T163Y) abolished only molybdate adsorption, exhibiting the specific adsorption of tungstate. The specificity of the displayed ModE mutant protein (T163Y) for tungstate was increased by approximately 9.31-fold compared to the displayed wild-type ModE protein at pHxa05.4. Therefore, the strategy of protein design and its cell surface display is effective for the molecular breeding of bioadsorbents with metal-specific adsorption ability based on a single species of microorganism without isolation from nature.

Construction of a novel selection system for endoglucanases exhibiting carbohydrate-binding modules optimized for biomass using yeast cell-surface engineering

To permit direct cellulose degradation and ethanol fermentation, Saccharomyces cerevisiae BY4741 (Δsed1) codisplaying 3 cellulases (Trichoderma reesei endoglucanase II [EG], T. reesei cellobiohydrolase II [CBH], and Aspergillus aculeatus β-glucosidase I [BG]) was constructed by yeast cell-surface engineering. The EG used in this study consists of a family 1 carbohydrate-binding module (CBM) and a catalytic module. A comparison with family 1 CBMs revealed conserved amino acid residues and flexible amino acid residues. The flexible amino acid residues were at positions 18, 23, 26, and 27, through which the degrading activity for various cellulose structures in each biomass may have been optimized. To select the optimal combination of CBMs of EGs, a yeast mixture with comprehensively mutated CBM was constructed. The mixture consisted of yeasts codisplaying EG with mutated CBMs, in which 4 flexible residues were comprehensively mutated, CBH, and BG. The yeast mixture was inoculated in selection medium with newspaper as the sole carbon source. The surviving yeast consisted of RTSH yeast (the mutant sequence of CBM: N18R, S23T, S26S, and T27H) and wild-type yeast (CBM was the original) in a ratio of 1:46. The mixture (1 RTSH yeast and 46 wild-type yeasts) had a fermentation activity that was 1.5-fold higher than that of wild-type yeast alone in the early phase of saccharification and fermentation, which indicates that the yeast mixture with comprehensively mutated CBM could be used to select the optimal combination of CBMs suitable for the cellulose of each biomass.

Design of a Novel Antimicrobial Peptide Activated by Virulent Proteases

Antimicrobial peptides are promising antibiotics as they possess strong antimicrobial activity and very broad spectra of activity. However, administration of an antibiotic with a very broad spectrum of activity disrupts normal microflora and increases the risks of other fatal infections. To solve the problem, we designed a novel antimicrobial peptide that is activated by virulent proteases of pathogenic organisms. We constructed a peptide composed of three domains, namely an antimicrobial peptide (lactoferricin) as the active center, a protective peptide (magainin intervening sequence) that suppresses antimicrobial activity, and a specific linker that joins these two components and is efficiently cleaved by virulent proteases. We utilized Candida albicans as a model organism that produces secreted aspartic proteases as a virulence attribute. We screened for a peptide sequence efficiently cleaved by secreted aspartic proteases isozymes and identified a GFIKAFPK peptide as the most favorable substrate. Subsequently, we chemically synthesized a peptide containing the GFIKAFPK sequence. The designed peptide possessed no antimicrobial activity until it was activated by secreted aspartic proteases isozymes. Furthermore, it demonstrated selective antimicrobial activity against C. albicans, but not against Saccharomyces cerevisiae. A designed peptide like the one described in this study may protect normal microflora, resulting in enhanced safety as a therapeutic.

Mutated intramolecular chaperones generate high-activity isomers of mature enzymes.

The propeptide of carboxypeptidase Y precursor (proCPY) acts as an intramolecular chaperone that ensures the correct folding of the mature CPY (mCPY). Here, to further characterize the folding mechanism mediated by the propeptide, folding analysis was performed using a yeast molecular display system. CPYs with mutated propeptides were successfully displayed on yeast cell surface, and the mature enzymes were purified by the selective cleavage of mutated propeptides. Measurement of the activity and kinetics of the displayed CPYs indicated that the propeptide mutation altered the catalytic efficiency of mCPY. Although the mature region of the wild-type and mutant CPYs had identical amino acid sequences, the mCPYs from the mutant proCPYs had higher catalytic efficiency than the wild-type. These results indicate that proteins with identical amino acid sequence can fold into isomeric proteins with conformational microchanges through mutated intramolecular chaperones.