Hygrolansamycins A-D, O-Heterocyclic Macrolides from Streptomyces sp. KCB17JA11.

Six ansamycin derivatives were isolated from the culture broth of Streptomyces sp. KCB17JA11, including four new hygrolansamycins A-D (1-4) and known congeners divergolide O (5) and hygrocin C (6). Compounds 1-5 featured an unusual six-membered O-heterocyclic moiety. The isolation workflow was guided by a Molecular Networking-based dereplication strategy. The structures of 1-4 were elucidated using NMR and HRESIMS experiments, and the absolute configuration was established by the Mosher's method. Compound 2 exhibited mild cytotoxicity against five cancer cell lines with IC50 values ranging from 24.60 ± 3.37 µM to 49.93 ± 4.52 µM.

Jejuketomycins A and B, polyketide glycosides with cancer cell migration inhibitory activity from Streptomyces sp. KCB15JA151.

Two new polyketide glycosides jejuketomycins A (1) and B (2), were isolated from a culture of Streptomyces sp. KCB15JA151. Their chemical structures including the absolute configurations were determined by detailed analyses of the NMR and HRMS data and ECD calculations and spectral data. Compounds 1 and 2 possess an unusual 6/6/8 tricyclic ring system. Biological evaluation with the wound healing assay and time-lapse cell tracking analysis revealed that compounds 1 and 2 have significant inhibitory activities against cancer cell migration with low cytotoxicity.

Reassortant Highly Pathogenic H5N6 Avian Influenza Virus Containing Low Pathogenic Viral Genes in a Local Live Poultry Market, Vietnam.

Sites of live poultry trade and marketing are hot spots for avian influenza virus (AIV) transmission. We conducted active surveillance at a local live poultry market (LPM) in northern Vietnamese provinces in December 2016. Feces samples from the market were collected and tested for AIV. A new reassorted AIV strain was isolated from female chickens, named A/chicken/Vietnam/AI-1606/2016 (H5N6), and was found to belong to group C of clade 2.3.4.4 H5N6 highly pathogenic (HP) AIVs. The neuraminidase gene belongs to the reassortant B type. The viral genome also contained polymerase basic 2 and polymerase acidic, which were most closely related to domestic-duck-origin low pathogenic AIVs in Japan (H3N8) and Mongolia (H4N6). The other six genes were most closely related to poultry-origin H5N6 HP AIVs in Vietnam and had over 97% sequence identity with human AIV isolate A/Guangzhou/39715/2014 (H5N6). The new reassorted AIV isolate A/chicken/Vietnam/AI-1606/2016 (H5N6) identified in this study exemplifies AIVs reassortment and evolution through contact among wild birds, poultry farms, and LPMs. Therefore, active surveillance of AIVs is necessary to prevent potential threats to human and animal health.

Functional Characterization of the mazEF Toxin-Antitoxin System in the Pathogenic Bacterium Agrobacterium tumefaciens.

Agrobacterium tumefaciens is a pathogen of various plants which transfers its own DNA (T-DNA) to the host plants. It is used for producing genetically modified plants with this ability. To control T-DNA transfer to the right place, toxin-antitoxin (TA) systems of A. tumefaciens were used to control the target site of transfer without any unintentional targeting. Here, we describe a toxin-antitoxin system, Atu0939 (mazE-at) and Atu0940 (mazF-at), in the chromosome of Agrobacterium tumefaciens. The toxin in the TA system has 33.3% identity and 45.5% similarity with MazF in Escherichia coli. The expression of MazF-at caused cell growth inhibition, while cells with MazF-at co-expressed with MazE-at grew normally. In vivo and in vitro assays revealed that MazF-at inhibited protein synthesis by decreasing the cellular mRNA stability. Moreover, the catalytic residue of MazF-at was determined to be the 24th glutamic acid using site-directed mutagenesis. From the results, we concluded that MazF-at is a type II toxin-antitoxin system and a ribosome-independent endoribonuclease. Here, we characterized a TA system in A. tumefaciens whose understanding might help to find its physiological function and to develop further applications.

Engineered Escherichia coli strains as platforms for biological production of isoprene.

Volatile compounds can be produced by fermentation from genetically engineered microorganisms. Escherichia coli strains are mainly used for isoprene production owing to their higher titers; however, this has thus far been confined to only strains BL21, BL21 (DE3), Rosetta, and BW25113. Here, we tested four groups of E. coli strains for improved isoprene production, including K-12 (DH5α, BW25113, W3110, MG1655, XL1-Blue, and JM109), B [Rosetta (DE3), BL21, and BL21 (DE3)], Crooks C, and Waksman W strains. The isoprene productivity of BL21 and MG1655 was remarkably higher than that of the others in 5-L fermentation, and scale-up fermentation (300 L) of BL21 was successfully performed. This system shows potential for biobased production of fuel and volatile compounds in industrial applications.

Production of Bio-Based Isoprene by the Mevalonate Pathway Cassette in Ralstonia eutropha.

Isoprene has the potential to replace some petroleum-based chemicals and can be produced through biological systems using renewable carbon sources. Ralstonia eutropha can produce value-added compounds, including intracellular polyhydroxyalkanoate (PHA) through fatty acid and lipid metabolism. In the present study, we engineered strains of R. eutropha H16 and examined the strains for isoprene production. We optimized codons of all the genes involved in isoprene synthesis by the mevalonate pathway and manipulated the promoter regions using pLac and pJ5 elements. Our results showed that isoprene productivity was higher using the J5 promoter (1.9 ± 0.24 µg/l) than when using the lac promoter (1.5 ± 0.2 µg/l). Additionally, the use of three J5 promoters was more efficient (3.8 ± 0.18 µg/l) for isoprene production than a one-promoter system, and could be scaled up to a 5-L batch-cultivation from a T-flask culture. Although the isoprene yield obtained in our study was insufficient to meet industrial demands, our study, for the first time, shows that R. eutropha can be modified for efficient isoprene production and lays the foundation for further optimization of the fermentation process.

Development of novel on-line capillary gas chromatography-based analysis method for volatile organic compounds produced by aerobic fermentation.

Many volatile compounds, such as isoprene, a precursor used in the synthesis of natural rubber, have been produced through fermentation using genetically engineered microorganisms. Despite this biotechnological success, measuring the concentrations of volatile compounds during fermentation is difficult because of their high volatility. In current systems, off-line analytical methods usually lead to product loss, whereas on-line methods raise the production cost due to the requirement of complex devices. Here, we developed a novel on-line gas chromatography (GC)-based system for analyzing the concentration of isoprene with the aim to minimize the cost and requirement for devices as compared to current strategies. In this system, a programmable logic controller is used to combine conventional GC with a syringe pump module (SPM) directly connected to the exhaust pipe of the fermentor, and isoprene-containing samples are continuously pumped from the SPM into the GC using an air cylinder recycle stream. We showed that this novel system enables isoprene analysis during fermentation with convenient equipment and without the requirement of an expensive desorption tube. Furthermore, this system may be extended to the detection of other volatile organic compounds in fermentation or chemical processes.

Development of a promising microbial platform for the production of dicarboxylic acids from biorenewable resources.

BACKGROUND: As a sustainable industrial process, the production of dicarboxylic acids (DCAs), used as precursors of polyamides, polyesters, perfumes, plasticizers, lubricants, and adhesives, from vegetable oil has continuously garnered interest. Although the yeast Candida tropicalis has been used as a host for DCA production, additional strains are continually investigated to meet productivity thresholds and industrial needs. In this regard, the yeast Wickerhamiella sorbophila, a potential candidate strain, has been screened. However, the lack of genetic and physiological information for this uncommon strain is an obstacle that merits further research. To overcome this limitation, we attempted to develop a method to facilitate genetic recombination in this strain and produce high amounts of DCAs from methyl laurate using engineered W. sorbophila. RESULTS: In the current study, we first developed efficient genetic engineering tools for the industrial application of W. sorbophila. To increase homologous recombination (HR) efficiency during transformation, the cell cycle of the yeast was synchronized to the S/G2 phase using hydroxyurea. The HR efficiency at POX1 and POX2 loci increased from 56.3% and 41.7%, respectively, to 97.9% in both cases. The original HR efficiency at URA3 and ADE2 loci was nearly 0% during the early stationary and logarithmic phases of growth, and increased to 4.8% and 25.6%, respectively. We used the developed tools to construct W. sorbophila UHP4, in which ß-oxidation was completely blocked. The strain produced 92.5 g/l of dodecanedioic acid (DDDA) from methyl laurate over 126 h in 5-l fed-batch fermentation, with a productivity of 0.83 g/l/h. CONCLUSIONS: Wickerhamiella sorbophila UHP4 produced more DDDA methyl laurate than C. tropicalis. Hence, we demonstrated that W. sorbophila is a powerful microbial platform for vegetable oil-based DCA production. In addition, by using the developed genetic engineering tools, this emerging yeast could be used for the production of a variety of fatty acid derivatives, such as fatty alcohols, fatty aldehydes, and ω-hydroxy fatty acids.

Enhancement of L-Threonine Production by Controlling Sequential Carbon-Nitrogen Ratios during Fermentation.

Controlling the residual glucose concentration is important for improving productivity in L-threonine fermentation. In this study, we developed a procedure to automatically control the feeding quantity of glucose solution as a function of ammonia-water consumption rate. The feeding ratio (RC/N) of glucose and ammonia water was predetermined via a stoichiometric approach, on the basis of glucose-ammonia water consumption rates. In a 5-L fermenter, 102 g/l L-threonine was obtained using our glucose-ammonia water combined feeding strategy, which was then successfully applied in a 500-L fermenter (89 g/l). Therefore, we conclude that an automatic combination feeding strategy is suitable for improving L-threonine production.