Search

Polyene macrolides, such as nystatin A1, amphotericin B, and NPP A1, belong to a large family of valuable antifungal polyketide compounds that are typically produced by soil actinomycetes. Previously, NPP B1, a novel NPP A1 derivative harboring a heptaene core structure, was generated by introducing two amino acid substitutions in the putative NADPH-binding motif of the enoyl reductase domain in module 5 of the NPP A1 polyketide synthase in Pseudonocardia autotrophica. This derivative showed superior antifungal activity to NPP A1. In this study, another novel derivative called NPP B2 was developed, which lacks a hydroxyl group at the C10 position by site-specific gene disruption of the P450 hydroxylase NppL. To stimulate the extremely low expression of the NPP B2 biosynthetic pathway genes, the 32-kb NPP-specific regulatory gene cluster was overexpressed via site-specific chromosomal integration. The extra copy of the six NPP-specific regulatory genes led to a significant increase in the NPP B2 yield from 0.19 to 7.67 mg/L, which is the highest level of NPP B2 production ever achieved by the P. autotrophica strain. Subsequent in vitro antifungal activity and toxicity studies indicated that NPP B2 exhibited similar antifungal activity but significantly lower hemolytic toxicity than NPP B1. These results suggest that an NPP biosynthetic pathway refactoring and overexpression of its pathway-specific regulatory genes is an efficient approach to stimulating the production of an extremely low-level metabolite, such as NPP B2 in a pathway-engineered rare actinomycete strain.

Pseudonocardia autotrophica was previously identified to produce a toxicity-reduced and solubility-improved disaccharide-containing anti-fungal compound belonging to the tetraene-family, Nystatin-like Pseudonocardia Polyene A1 (NPP A1). Subsequently NPP B1, a novel derivative harboring a heptaene core structure, was produced by a pathway-engineered Pseudonocardia strain through inactivation of the specific enoly reductase gene domain in the NPP biosynthetic gene cluster. Although in vitro and in vivo efficacy and toxicity studies indicate that NPP B1 is a promising lead antifungal compound, further improvement is required to increase the extremely low production yield in the pathway-engineered strain. To overcome this challenge, we performed the N-methyl-N′-nitro-N-nitrosoguanidine (NTG) iterative random mutagenesis, followed by zone-of-inhibition agar plug assay. After three rounds of the mutagenesis-and-screening protocol, the production yield of NPP B1 increased to 6.25 mg/L, which is more than an eightfold increase compared to the parental strain. The qRT-PCR analysis revealed that transcripts of the NPP B1 biosynthetic genes were increased in the mutant strain. Interestingly, an endogenous 125-kb plasmid was found to be eliminated through this mutagenesis. To further improve the NPP B1 production yield, the 32-kb NPP-specific regulatory gene cluster was cloned and overexpressed in the mutant strain. The chromosomal integration of the extra copy of the six NPP-specific regulatory genes led to an additional increase of NPP B1 yield to 31.6 mg/L, which is the highest production level of NPP B1 ever achieved by P. autotrophica strains. These results suggest that a synergistic combination of both the traditional and genetic strain improvement approaches is a very efficient strategy to stimulate the production of an extremely low-level metabolite (such as NPP B1) in a pathway-engineered rare actinomycetes strain.

Polyene macrolides such as nystatin A1 and amphotericin B belong to a large family of very valuable antifungal polyketide compounds typically produced by soil actinomycetes. Recently, nystatin-like Pseudonocardia polyene (NPP) A1 has been identified as a unique disaccharide-containing tetraene antifungal macrolide produced by Pseudonocardia autotrophica. Despite its significantly increased water solubility and decreased hemolytic activity, its antifungal activity remains limited compared with that of nystatin A1. In this study, we developed NPP B1, a novel NPP A1 derivative harboring a heptaene core structure, by introducing two amino acid substitutions in the putative NADPH-binding motif of the enoyl reductase domain in module 5 of the NPP A1 polyketide synthase NppC. The low level NPP B1 production yield was successfully improved by eliminating the native plasmid encoding a polyketide biosynthetic gene cluster present in P. autotrophica. In vitro and in vivo antifungal activity and toxicity studies indicated that NPP B1 exhibited comparable antifungal activity against Candida albicans and was less toxic than the most potent heptaene antifungal, amphotericin B. Moreover, NPP B1 showed improved pharmacokinetic parameters compared to those of amphotericin B, suggesting that NPP B1 could be a promising candidate for development into a pharmacokinetically improved and less-toxic polyene antifungal antibiotic.

This paper describes loss of coolant accident (LOCA) analyses of the Supercritical-pressure Water-Cooled Fast Reactor (Super Fast Reactor). The features of the Super Fast Reactor are high power density and downward flow cooled fuel channels for the improvement of the economic potential of the Super Fast Reactor with high outlet steam temperature. The LOCA induces large pressure and coolant density change in the core. This change influences the flow distribution among the downward flow parallel channels. It will affect the safety of the Super Fast Reactor. LOCA analysis of Super Fast Reactor is important to understand the safety features of the Super Fast Reactor. Keeping the flow rate in the core is important for the safety of the Super Fast Reactor. In LOCA, it is difficult to maintain an adequate flow rate due to the once-through coolant cycle and the downward flow cooled fuel assemblies. Therefore, the early actuation of the Automatic Depressurization System (ADS) and reduction of the maximum linear he...

The practical feasibility of 126Sn transmutation in spallation targets of accelerator-driven systems was evaluated from the viewpoints of accumulation of radioactive spallation products and neutron production as well as transmutation amount of 126Sn. A cylindrical liquid 126Sn target whose length depends on proton beam energy was described, based on a Pb-Bi target design of accelerator-driven system being developed in JAEA. A proton beam of 1.5 GeV-20mA was estimated to give the transmutation rate of 126Sn 6.4 kg/yr, which corresponds to the amount of 126Sn annually discharged in 27 LWRs of 1GWt and 33 GWd/THM. The equilibrium radioactivity of spallation products would reach 9% of that of 126Sn transmuted in the spallation target, and the equilibrium toxicity would be just 3%. Some parametric analyses showed that the effective half-life of 126Sn could be reduced through a proper reduction of the target size. The 126Sn target was calculated to produce 40 neutrons per proton of 1.5 GeV and give a neutron sp...

A subcritical reactor for TRU (Transuranics) transmutation, HYPER (HYbrid Power Extraction Reactor), is under development at KAERI (Korea Atomic Energy Research Institute). For the HYPER system, a pyrochemical process is being considered for fuel reprocessing, due to its own characteristics of nuclear proliferation resistance. Separated from the partitioning process, the fuel contains not only TRU but also the considerable percentages of impurities such as uranium and lanthanides. In our previous studies, the eigen mode calculation had been performed without the external source effects, for the purpose of analyzing the fuel composition effects on the HYPER core characteristics. And the results had shown that the dynamic behavior of the HYPER system becomes unstable as the amount of impurities increases. In this paper, the external source neutron effects on the core characteristics sensitivity to the fuel composition were investigated. Assuming various recovery factors of uranium and lanthanides, some dyna...