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No abstract

"Candidatus Accumulibacter phosphatis" is considered a polyphosphate-accumulating organism (PAO) though it has not been isolated yet. To reveal the denitrification ability of this organism, we first concentrated this organism by flow cytometric sorting following fluorescence in situ hybridization (FISH) using specific probes for this organism. The purity of the target cells was about 97% of total cell count in the sorted sample. The PCR amplification of the nitrite reductase genes (nirK and nirS) from unsorted and sorted cells was performed. Although nirK and nirS were amplified from unsorted cells, only nirS was detected from sorted cells, indicating that "Ca. Accumulibacter phosphatis" has nirS. Furthermore, nirS fragments were cloned from unsorted (Ba clone library) and sorted (Bd clone library) cells and classified by restriction fragment length polymorphism analysis. The most dominant clone in clone library Ba, which represented 62% of the total number of clones, was not found in clone library Bd. In contrast, the most dominant clone in clone library Bd, which represented 59% of the total number of clones, represented only 2% of the total number of clones in clone library Ba, indicating that this clone could be that of "Ca. Accumulibacter phosphatis." The sequence of this nirS clone exhibited less than 90% similarity to the sequences of known denitrifying bacteria in the database. The recovery of the nirS genes makes it likely that "Ca. Accumulibacter phosphatis" behaves as a denitrifying PAO capable of utilizing nitrite instead of oxygen as an electron acceptor for phosphorus uptake.

Since it was recently reported that an antibody for proprotein convertase subtilisin/kexin type 9 (PCSK9) reduces the risk of cardiovascular events in a clinical context, PCSK9 inhibition is thought to be an attractive therapy for dyslipidemia. In the present study, we created a novel small biologic alternative to PCSK9 antibodies called DS-9001a, comprising an albumin binding domain fused to an artificial lipocalin mutein (ABD-fused Anticalin protein), which can be produced by a microbial production system. DS-9001a strongly interfered with PCSK9 binding to low-density-lipoprotein receptor (LDL-R) and PCSK9-mediated degradation of LDL-R. In cynomolgus monkeys, single DS-9001a administration significantly reduced the serum LDL-C level up to 21 days (62.4% reduction at the maximum). Moreover, DS-9001a reduced plasma non-high-density-lipoprotein cholesterol and oxidized LDL levels, and their further reductions were observed when atorvastatin and DS-9001a were administered in combination in human cholesteryl ester transfer protein/ApoB double transgenic mice. Additionally, their reductions on the combination of atorvastatin and DS-9001a were more pronounced than those on the combination of atorvastatin and anacetrapib. Besides its favorable pharmacologic profile, DS-9001a has a lower molecular weight (about 22 kDa), yielding a high stoichiometric drug concentration that might result in a smaller administration volume than that in existing antibody therapy. Since bacterial production systems are viewed as more suited to mass production at low cost, DS-9001a may provide a new therapeutic option to treat patients with dyslipidemia. In addition, considering the growing demand for antibody-like drugs, ABD-fused Anticalin proteins could represent a promising new class of small biologic molecules.

No abstract

The fungus Thelonectria discophora SANK 18292 produces the iminosugar nectrisine, which has a nitrogen-containing heterocyclic 5-membered ring and acts as a glycosidase inhibitor. In our previous study, an oxidase (designated NecC) that converts 4-amino-4-deoxyarabinitol to nectrisine was purified from T. discophora cultures. However, the genes required for nectrisine biosynthesis remained unclear. In this study, the nectrisine biosynthetic gene cluster in T. discophora was identified from the contiguous genome sequence around the necC gene. Gene disruption and complementation studies and heterologous expression of the gene showed that necA, necB, and necC could be involved in nectrisine biosynthesis, during which amination, dephosphorylation, and oxidation occur. It was also demonstrated that nectrisine could be produced by recombinant Escherichia coli coexpressing the necA, necB, and necC genes. These findings provide the foundation to develop a bacterial production system for nectrisine or its intermediates through genetic engineering. IMPORTANCEIminosugars might have great therapeutic potential for treatment of many diseases. However, information on the genes for their biosynthesis is limited. In this study, we report the identification of genes required for biosynthesis of the iminosugar nectrisine in Thelonectria discophora SANK 18292, which was verified by disruption, complementation, and heterologous expression of the genes involved. We also demonstrate heterologous production of nectrisine by recombinant E. coli, toward developing an efficient production system for nectrisine or its intermediates through genetic engineering.

A fungus, Thelonectria discophora SANK 18292 (JCM 30947), produces nectrisine that has a nitrogen-containing heterocyclic 5-membered ring acting as a glycosidase inhibitor. Our previous study showed the possibility that 4-amino-4-deoxyarabinitol was enzymatically converted to nectrisine but the enzyme was not known. In order to characterize the enzyme, which is designated as NecC, it was purified from the fungus using ammonium sulfate precipitation and anion exchange chromatography. Liquid chromatography-tandem mass spectrometry analysis of NecC tryptic digests revealed partial NecC protein sequences. Subsequently, the partial DNA fragments were amplified by polymerase chain reaction with degenerate oligonucleotide primers and cloned. Then, necC complete genomic DNA was cloned by screening a genomic library of the fungus. Recombinant NecC also had NecC enzymatic activity, thus providing verification for the necC gene. NecC presumably belonged to the family of glucose methanol choline oxidoreductases, forming oligomers ranging approximately from 8 mer to 16 mer based on the results of native PAGE, and was also found to have a melting temperature of 57 °C, an optimal reaction condition of pH 7 at 30 °C, an activity inhibited by Cu2+ or ethylenediaminetetraacetic acid, and 4-amino-4-deoxyarabinitol as its preferred substrate. It was also indicated that not nectrisine but 4-amino-4-deoxyarabinitol was mainly extracted from the mycelium, and then was converted to nectrisine by the enzyme NecC in vitro. We believe that these findings are helpful to establish a nectrisine manufacturing process at large scale with the fungus.Electronic supplementary materialThe online version of this article (doi:10.1186/s13568-016-0176-1) contains supplementary material, which is available to authorized users.

Some monoclonal antibodies undergo liquid–liquid phase separation owing to self-attractive associations involving electrostatic and other soft interactions, thereby rendering monoclonal antibodies unsuitable as therapeutics. To mitigate the phase separation, formulation optimization is often performed. However, this is sometimes unsuccessful because of the limited time for the development of therapeutic antibodies. Thus, protein mutations with appropriate design are required. In this report, we describe a case study involving the design of mutants of negatively charged surface residues to reduce liquid–liquid phase separation propensity. Physicochemical analysis of the resulting mutants demonstrated the mutual correlation between the sign of second virial coefficient B 2 , the Fab dipole moment, and the reduction of liquid–liquid phase separation propensity. Moreover, both the magnitude and direction of the dipole moment appeared to be essential for liquid–liquid phase separation propensity, where electrostatic interaction was the dominant mechanism. These findings could contribute to a better design of mutants with reduced liquid–liquid phase separation propensity and improved drug-like biophysical properties.