Optimisation of hypocrellin production in Shiraia-like fungi via genetic modification involving a transcription factor gene and a putative monooxygenase gene.

Shiraia-like fungi, which are rare parasitic fungi found around bamboo, play an important role in traditional medicine. Their main active component, hypocrellin, is widely used in medicine, food, and cosmetics. By comparing strains with different hypocrellin yields, we identified a transcription factor (SbTF) in the hypocrellin biosynthesis pathway. SbTF from high-yielding zzz816 and low-yielding CNUCC C72 differed in its protein structure. Subsequently, SbTF from high-yielding zzz816 was overexpressed in several strains. This stabilised the yield in zzz816 and significantly increased the yield in low-yielding CNUCC C72. Comparing downstream non-essential genes between wild type and SbTF-overexpressing CNUCC C72 showed that SbMNF was significantly up-regulated. Therefore, it was selected for further study. SbMNF overexpression increased the hypocrellin yield in low-yielding CNUCC C72 and altered the composition of compounds in high-yielding CNUCC 1353PR and zzz816. This involved an increased elsinochrome C yield in CNUCC 1353PR and an increased hypocrellin B yield in zzz816 (by 2 and 70.3 times that in the corresponding wild type, respectively). This study is the first to alter hypocrellin synthesis to alter the levels of one bioactive agent compared to another. The results provide new insights regarding genetic modification and will help to optimise fungal fermentation.

Discovery of myricetin as an inhibitor against Streptococcus mutans and an anti-adhesion approach to biofilm formation.

Streptococcus mutans (S. mutans) are cariogenic microorganisms. Sortase A (SrtA) is a transpeptidase that attaches Pac to the cell surface. The biofilm formation of S. mutans is promoted by SrtA regulated Pac. Myricetin (Myr) has a variety of pharmacological properties, including inhibiting SrtA activity of Staphylococcus aureus. The purpose of this research was to investigate the inhibitory effect of Myr on SrtA of S. mutans and its subsequent influence on the biofilm formation. Here, Myr was discovered as a potent inhibitor of S. mutans SrtA, with an IC50 of 48.66 ± 1.48 µM, which was lower than the minimum inhibitory concentration (MIC) of 512 ug/mL. Additionally, immunoblot and biofilm assays demonstrated that Myr at a sub-MIC level could reduce adhesion and biofilm formation of S. mutans. The reduction of biofilm was possibly caused by the decreased amount of Pac on the cells' surface by releasing Pac into the medium via inhibiting SrtA activity. Molecular dynamics simulations and mutagenesis assays suggested that Met123, Ile191, and Arg213 of SrtA were pivotal for the interaction of SrtA and Myr. Our findings indicate that Myr is a promising candidate for the control of dental caries by modulating Pac-involved adhesive mechanisms without developing drug resistance to S.mutans.

Total Syntheses of Resin Glycosides Murucoidins IV and V.

Murucoidins IV and V, two bioactive resin glycosides with complex yet similar structures isolated from the morning glory family, were synthesized in a convergent manner. All of the glycosylations in these syntheses including the key [3 + 2] coupling were achieved by our recently developed interrupted Pummerer reaction mediated (IPRm) glycosylations. The broad functional group compatibility of IPRm glycosylation allowed us to employ a latent-active concept and a single-pot transient protection-glycosylation-deprotection strategy which significantly improved the global synthetic efficiency.

Practical synthesis of active O-2-(2-propylsulfinyl)benzyl (OPSB) glycosides via a catalytic and metal free oxidation of latent O-2-(2-propylthiol)benzyl (OPTB) glycosides.

A catalytic and metal free sulfoxidation of O-2-(2-propylthiol)benzyl (OPTB) glycosides to O-2-(2-propylsulfinyl)benzyl (OPSB) glycosides has been developed by introducing NOBF4 as catalyst, oxygen as terminal oxidant and TBAB as additive. Wide variety of OPTB glycosides were efficiently oxidized without observation of over oxidation. The allowance of large scale synthesis, easy operation and purification highlighted its practical application in construction of complex oligosaccharides and glycoconjugates employing interrupted Pummerer reaction mediated glycosylation strategy.

Interrupted Pummerer Reaction in Latent-Active Glycosylation: Glycosyl Donors with a Recyclable and Regenerative Leaving Group.

Latent O-glycosides, 2-(2-propylthiol)benzyl (PTB) glycosides, were converted into the corresponding active glycosyl donors, 2-(2-propylsulfinyl)benzyl (PSB) glycosides, by a simple and efficient oxidation. Treatment of the PSB donor and various acceptors with triflic anhydride provided the desired glycosides in good to excellent yields. The leaving group, which was activated by an interrupted Pummerer reaction, can be recycled (PSB-OH) and regenerated as the precursor (PTB-OH). A natural hepatoprotective glycoside, leonoside F, was efficiently synthesized in a convergent [3+1] manner with this newly developed method. The present total synthesis also led to a structural revision of this phenylethanoid glycoside.

Influence of Pseudomonas aeruginosa quorum sensing signal molecule N-(3-oxododecanoyl) homoserine lactone on mast cells.

Quorum sensing system is a cell-to-cell communication system that plays a pivotal role in virulence expression in bacteria. Recent advances have demonstrated that the Pseudomonas aeruginosa quorum sensing molecule, N-3-oxododecanoyl homoserine lactone (3OC(12)-HSL), exerts effects on mammalian cells and modulates host immune response. Mast cells (MCs) are strategically located in the tissues that are constantly exposed to external stimulus. Therefore, it is very much possible that 3OC(12)-HSL may interact with MCs. Little is known, however, about specific effects of 3OC(12)-HSL on MCs. To address this, we investigated the influence of 3OC(12)-HSL on cell viability, apoptosis, intracellular calcium and cytokine release in MCs. We found that at high concentrations (100 microM), 3OC(12)-HSL inhibited proliferation and induced apoptosis in P815. The 3OC(12)-HSL treatment significantly increased intracellular calcium release in both P815 and HMC-1. We also observed that 3OC(12)-HSL-induced histamine release and degranulation in HMC-1 cells. Furthermore, 3OC(12)-HSL-induced IL-6 production at lower concentrations (6.25-12.5 microM) but steadily reduced IL-6 production at high concentration (50-100 muM). These data demonstrate that P. aeruginosa 3OC(12)-HSL affects MCs function.

Enzymatic synthesis of a CCK-4 tripeptide fragment.

OBJECTIVE: To synthesize a tripeptide derivative Phac-Met-Asp(OMe)-Phe -NH2, which is a fragment of the gastrin C-terminal tetrapeptide CCK-4, by enzymatic reaction. METHODS: Three free enzymes, alpha-chymotrypsin, papain and thermolysin from acyl donor Phac-Met-OCam was involved in three steps. The choice of appropriate enzymes and solvents was selected. RESULTS: All enzymatic reactions were obtained in reasonable yields(63%-92%). FAB-MS and FD-MS verified the correct molecular mass of the peptides. CONCLUSION: Studies on the alpha-chymotrypsin catalyzed coupling reaction between Phac-Met-OCam and H-Asp(OMe)2 have focused on the low water content media. By papain catalyzed saponification of Phac-Met-Asp(OMe)2, alpha-methyl ester of aspartic acid is selectively hydrolyzed to retain beta-methyl ester, and Phac-Met-Asp(OMe)-OH and H-Phe-NH2 can be coupled efficiently by thermolysin.

Synthesis of cholecystokinin peptide CCK-4 exclusively by enzymatic methods.

The synthesis of CCK-4 (H-Trp-Met-Asp-Phe-NH2) by using enzymes exclusively was described. As protection group for the amino group we used the Phenylacetyl group (Phac) which had been cleaved at the end of the synthesis with Penicillin G Amidase (PGA) without affecting the peptide bonds. Thus, beginning with Phac-Trp-OH we had successfully synthesized the target peptide with following 4 enzymes, alpha-Chymotrypsin, Papain, Thermolysin and PGA in four reaction steps. All reactions were carried out in aqueous buffer in reasonable yields (> 65%). FAB-MS or FD-MS verified the correct molecular mass of all peptides.