Anthranilate derivatives have been used as flavoring and fragrant agents for a long time. Recently, these compounds are gaining attention due to new biological functions including antinociceptive and analgesic activities. Three anthranilate derivatives, N-methylanthranilate, methyl anthranilate, and methyl N-methylanthranilate were synthesized using metabolically engineered stains of Escherichia coli. NMT encoding N-methyltransferase from Ruta graveolens, AMAT encoding anthraniloyl-coenzyme A (CoA):methanol acyltransferase from Vitis labrusca, and pqsA encoding anthranilate coenzyme A ligase from Pseudomonas aeruginosa were cloned and E. coli strains harboring these genes were used to synthesize the three desired compounds. E. coli mutants (metJ, trpD, tyrR mutants), which provide more anthranilate and/or S-adenosyl methionine, were used to increase the production of the synthesized compounds. MS/MS analysis was used to determine the structure of the products. Approximately, $185.3{\mu}M$ N-methylanthranilate and $95.2{\mu}M$ methyl N-methylanthranilate were synthesized. This is the first report about the synthesis of anthranilate derivatives in E. coli.

Synthetic biology builds programmed biological systems for a wide range of purposes such as improving human health, remedying the environment, and boosting the production of valuable chemical substances. In recent years, the rapid development of synthetic biology has enabled synthetic bacterium-based diagnoses and therapeutics superior to traditional methodologies by engaging bacterial sensing of and response to environmental signals inherent in these complex biological systems. Biosynthetic systems have opened a new avenue of disease diagnosis and treatment. In this review, we introduce designed synthetic bacterial systems acting as living therapeutics in the diagnosis and treatment of several diseases. We also discuss the safety and robustness of genetically modified synthetic bacteria inside the human body.

A series of thienopyrimidine compounds (6Aa-g and 6Ba-d) were synthesized and characterized by NMR spectroscopy and mass spectrometry. These compounds (6Aa-g and 6Ba-d) potently inhibited STAT3 expression induced by IL-6 in a dose-dependent manner with $IC_{50}$ values of $5.73-0.32{\mu}M$. Among the prepared thienopyrimidine derivatives, 6Aa, 6Ab, 6Ba and 6Bc significantly suppressed the phosphorylation of STAT3 and ERK1/2 stimulated by IL-6 in Hep3B cells. Furthermore, the synthesized compounds might be useful remedies for the treatment of inflammatory diseases by inhibiting the action of IL-6.

Farm animals such as piglets are often affected by environmental stress, which can disturb the gut ecosystem. Antibiotics were commonly used to prevent diarrhea in weaned piglets, but this was banned by the European Union due to the development of antibiotic resistance. However, the use of probiotics instead of antibiotics may reduce the risk posed by pathogenic microorganisms and reduce the incidence of gastrointestinal diseases. Therefore, this study was conducted to investigate the effects of Lactobacillus casei Zhang on the mechanical barrier and immune function of early-weaned piglets infected using Escherichia coli K88 based on histomorphology and immunology. Fourteen-day-old weaned piglets were divided into a control group and experimental groups that were fed L. casei Zhang and infected with E. coli K88 with or without prefeeding and/or postfeeding of L. casei Zhang. The L. casei Zhang dose used was $10^7CFU/g$ diet. Jejunum segments were obtained before histological, immunohistochemical, and western blot analyses were performed. In addition, the relative mRNA expression of toll receptors and cytokines was measured. Piglets fed L. casei Zhang showed significantly increased jejunum villus height, villus height-crypt depth ratio, muscle thickness, and expression of proliferating cell nuclear antigen and tight junction proteins ZO-1 and occludin. The use of L. casei Zhang effectively reduced intestinal inflammation after infection. We found that L. casei Zhang feeding prevented the jejunum damage induced by E. coli K88, suggesting that it may be a potential alternative to antibiotics for preventing diarrhea in early-weaned piglets.

Brewing with buckwheat as an ingredient has been proven to be successful in several previous studies. However, few studies have focused on the effects of buckwheat on the rutin content and antioxidant activity of beer. In order to develop a lager beer with high rutin content and desirable sensory characteristics, tartary buckwheat malt was used as a brewing adjunct. The results showed that the rutin-degrading enzyme was the key factor affecting the rutin content in the wort and beer. Compared to beer made using the common mashing method, the rutin content in the buckwheat beers produced using an improved mashing method was approximately 60 times higher. The total flavonoid contents in buckwheat beers also depended strongly on the mashing methods, ranging from 530.75 to 1,704.68 mg QE/l. The rutin-rich beers also showed better oxidative stability during forced-aging. Meanwhile, the buckwheat beers were found to be acceptable in terms of the main quality attributes, flavor, and taste.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)-based pathogen identification relies on the ribosomal protein spectra provided in the proprietary database. Although these mass spectra can discern various pathogens at species level, the spectra-based method still has limitations in identifying closely-related microbial species. In this study, to overcome the limits of the current MALDI-TOF MS identification method using ribosomal protein spectra, we applied MALDI-TOF MS of low-mass profiling to the identification of two genetically related Bacillus species, the food-borne pathogen Bacillus cereus, and the insect pathogen Bacillus thuringiensis. The mass spectra of small molecules from 17 type strains of two bacilli were compared to the morphological, biochemical, and genetic identification methods of pathogens. The specific mass peaks in the low-mass range (m/z 500-3,000) successfully identified various closely-related strains belonging to these two reference species. The intensity profiles of the MALDI-TOF mass spectra clearly revealed the differences between the two genetically-related species at strain level. We suggest that small molecules with low molecular weight, 714.2 and 906.5 m/z can be potential mass biomarkers used for reliable identification of B. cereus and B. thuringiensis.

Monascus purpureus recombinant mppC and mpp7 knockout strains were subjected to extractive fermentation in the context of azaphilone pigment production. Inclusion of Diaion HP-20 resin resulted in the selective production of unreduced azaphilone congeners, in addition to the early intermediate FK17-P2a, from ${\Delta}mppC$ and ${\Delta}mpp7$ strains that would otherwise mainly produce reduced congeners. Structural determination of two novel unreduced azaphilones from the ${\Delta}mpp7$ strain was accomplished. The unreduced azaphilone compound was converted into the cognate reduced congener in recombinant M. purpureus strains, demonstrating its intermediate role in azaphilone biosynthesis. This study demonstrates the possibility that extractive fermentation with Diaion HP-20 resin can be used to obtain cryptic azaphilone metabolites.

Bioethanol has attracted much attention in recent decades as a sustainable and environmentally friendly alternative energy source. In this study, we compared the production of bioethanol by Candida molischiana and Saccharomyces cerevisiae at different initial concentrations of cellobiose and glucose. The results showed that C. molischiana can utilize both glucose and cellobiose, whereas S. cerevisiae can only utilize glucose. The ethanol yields were 43-51% from different initial concentrations of carbon source. In addition, different concentrations of microcrystalline cellulose (Avicel) were directly converted to ethanol by a combination of Trichoderma reesei and two yeasts. Cellulose was first hydrolyzed by a fully enzymatic saccharification process using T. reesei cellulases, and the reducing sugars and glucose produced during the process were further used as carbon source for bioethanol production by C. molischiana or S. cerevisiae. Sequential culture of T. reesei and two yeasts revealed that C. molischiana was more efficient for bioconversion of sugars to ethanol than S. cerevisiae. When 20 g/l Avicel was used as a carbon source, the maximum reducing sugar, glucose, and ethanol yields were 42%, 26%, and 20%, respectively. The maximum concentrations of reducing sugar, glucose, and ethanol were 10.9, 8.57, and 5.95 g/l, respectively, at 120 h by the combination of T. reesei and C. molischiana from 50 g/l Avicel.

Magnetic $Ni_{0.7}Co_{0.3}Fe_2O_4$ nanoparticles that were prepared via the rapid combustion process were functionalized and modified to obtain magnetic $Ni_{0.7}Co_{0.3}Fe_2O_4@SiO_2-CHO$ nanocomposites, on which penicillin G acylase (PGA) was covalently immobilized. Selections of immobilization concentration and time of fixation were explored. Catalytic performance of immobilized PGA was characterized. The free PGA had greatest activity at pH 8.0 and $45^{\circ}C$ while immobilized PGA's activities peaked at pH 7.5 and $45^{\circ}C$. Immobilized PGA had better thermal stability than free PGA at the range of $30-50^{\circ}C$ for different time intervals. The activity of free PGA would be 0 and that of immobilized PGA still retained some activities at $60^{\circ}C$ after 2 h. $V_{max}$ and $K_m$ of immobilized PGA were 1.55 mol/min and 0.15 mol/l, respectively. Free PGA's $V_{max}$ and $K_m$ separately were 0.74 mol/min and 0.028 mol/l. Immobilized PGA displayed more than 50% activity after 10 successive cycles. We concluded that immobilized PGA with magnetic $Ni_{0.7}Co_{0.3}Fe_2O_4@SiO_2-CHO$ nanocomposites could become a novel example for the immobilization of other amidohydrolases.

Current strategies of strain improvement processes are mainly focused on enhancing the synthetic pathways of the products. However, excessive metabolic flux often creates metabolic imbalances, which lead to growth retardation and ultimately limit the yield of the product. To solve this problem, we applied a dynamic regulation strategy to produce $\text\tiny{L}$-phenylalanine ($\text\tiny{L}$-Phe) in Escherichia coli. First, we constructed a series of Phe-induced promoters that exhibited different strengths through modification of the promoter region of tyrP. Then, two engineered promoters were separately introduced into a Phe-producing strain xllp1 to dynamically control the expression level of one pathway enzyme AroK. Batch fermentation results of the strain xllp3 showed that the titer of Phe reached 61.3 g/l at 48 h, representing a titer of 1.36-fold of the strain xllp1 (45.0 g/l). Moreover, the $\text\tiny{L}$-Phe yields on glucose of xllp3 (0.22 g/g) were also greatly improved, with an increase of 1.22-fold in comparison with the xllp1 (0.18 g/g). In summary, we successfully improved the titer of Phe by using dynamic regulation of one key enzyme and this strategy can be applied for improving the performance of strains producing other aromatic amino acids and derived compounds.

Gamma-aminobutyric acid (GABA)-producing strains were isolated from four edible insects and subjected to 16S rRNA sequence analysis. Among the four GABA-producing bacteria, Enterococcus avium JS-N6B4 exhibited the highest GABA-production, while cultivation temperature, initial pH, aerobic condition, and mono-sodium glutamate (MSG) feeding were found to be the key factors affecting GABA production rate. The culture condition was optimized in terms of glucose, yeast extract, and MSG concentrations using response surface methodology (RSM). GABA production up to 16.64 g/l was obtained under the conditions of 7 g/l glucose, 45 g/l yeast extract, and 62 g/l MSG through the optimization of medium composition by RSM. Experimental GABA production was 13.68 g/l, which was close to the predicted value (16.64 g/l) calculated from the analysis of variance, and 2.79-fold higher than the production achieved with basic medium. Therefore, GABA-producing strains may help improve the GABA production in edible insects, and provide a new approach to the use of edible insects as effective food biomaterials.

Lipases are industrial enzymes that catalyze both triglyceride hydrolysis and ester synthesis. The overexpression of lipase genes is considered one of the best approaches to increase the enzymatic production for industrial applications. Subfamily I.2. lipases require a chaperone or foldase in order to become a fully-activated enzyme. The goal of this research was to isolate, clone, and co-express genes that encode lipase and foldase from Burkholderia territorii GP3, a lipolytic bacterial isolate obtained from Mount Papandayan soil via growth on Soil Extract Rhodamine Agar. Genes that encode for lipase (lipBT) and foldase (lifBT) were successfully cloned from this isolate and co-expressed in the E. coli BL21 background. The highest expression was shown in E. coli BL21 (DE3) pLysS, using pET15b expression vector. LipBT was particulary unique as it showed highest activity with optimum temperature of $80^{\circ}C$ at pH 11.0. The optimum substrate for enzyme activity was $C_{10}$, which is highly stable in methanol solvent. The enzyme was strongly activated by $Ca^{2+}$, $Mg^{2+}$, and strongly inhibited by $Fe^{2+}$ and $Zn^{2+}$. In addition, the enzyme was stable and compatible in non-ionic surfactant, and was strongly incompatible in ionic surfactant.

Chlorella spp. are green algae that are found across wide-ranging habitats from deserts to arctic regions, with various strains having adapted to survive under diverse environmental conditions. In this study, two novel Chlorella strains (ABC-002, ABC-008) were isolated from a freshwater lake in South Korea during the winter season and examined for possible use in the biofuel production process. The comparison of ABC-002 and ABC-008 strains with Chlorella vulgaris UTEX265 under two different temperatures ($10^{\circ}C$, $25^{\circ}C$) revealed their cold-tolerant phenotypes as well as high biomass yields. The maximum quantum yields of UTEX25, ABC-002, and ABC-008 at $10^{\circ}C$ were 0.5594, 0.6747, and 0.7150, respectively, providing evidence of the relatively higher cold-resistance capabilities of these two strains. Furthermore, both the biomass yields and lipid content of the two novel strains were found to be higher than those of UTEX265; the overall lipid productivities of ABC-002 and ABC-008 were 1.7 ~ 2.8 fold and 1.6 ~ 4.2 fold higher compared to that of UTEX265, respectively. Thus, the high biomass and lipid productivity over a wide range of temperatures indicate that C. vulgaris ABC-002 and ABC-008 are promising candidates for applications in biofuel productions via outdoor biomass cultivation.

Non-typhoidal Salmonella (NTS) is one of the most frequent causes of bacterial foodborne illnesses. Considering that the main reservoir of NTS is the intestinal tract of livestock, foods of animal origin are regarded as the main vehicles of Salmonella infection. In particular, poultry colonized with Salmonella Typhimurium (S. Typhimurium), a dominant serotype responsible for human infections, do not exhibit overt signs and symptoms, thereby posing a potential health risk to humans. In this study, comparative genomics approaches were applied to two S. Typhimurium strains, ST1539 and ST1120, isolated from a duck slaughterhouse and a pig farm, respectively, to characterize their virulence and antimicrobial resistance-associated genomic determinants. ST1539 containing a chromosome (4,905,039 bp; 4,403 CDSs) and a plasmid (93,876 bp; 96 CDSs) was phylogenetically distinct from other S. Typhimurium strains such as ST1120 and LT2. Compared to the ST1120 genome (previously deposited in GenBank; CP021909.1 and CP021910.1), ST1539 possesses more virulence determinants, including ST64B prophage, plasmid spv operon encoding virulence factors, genes encoding SseJ effector, Rck invasin, and biofilm-forming factors (bcf operon and pefAB). In accordance with the in silico prediction, ST1539 exhibited higher cytotoxicity against epithelial cells, better survival inside macrophage cells, and faster mice-killing activity than ST1120. However, ST1539 showed less resistance against antibiotics than ST1120, which may be attributed to the multiple resistanceassociated genes in the ST1120 chromosome. The accumulation of comparative genomics data on S. Typhimurium isolates from livestock would enrich our understanding of strategies Salmonella employs to adapt to diverse host animals.

It is well known that iron is critical for bacterial growth and pathogenic virulence. Due to chemical similarity, $Ga^{3+}$ competes with $Fe^{3+}$ for binding to compounds that usually bind $Fe^{3+}$, thereby interfering with various essential biological reactions. In our present study, gallium(III) nitrate [$Ga(NO_3)_3$] could repress the growth of V. splendidus Vs without complete inhibition. In the presence of $Ga(NO_3)_3$, the secretion of homogentisic acid-melanin (HGA-melanin) in V. splendidus Vs cells could be increased by 4.8-fold, compared to that in the absence of $Ga(NO_3)_3$. HGA-melanin possessed the ability to reduce $Fe^{3+}$ to $Fe^{2+}$. In addition, HGA-melanin increased the mRNA levels of feoA and feoB, genes coding Fe2+ transport system proteins to 1.86- and 6.1-fold, respectively, and promoted bacterial growth to 139.2%. Similarly, the mRNA expression of feoA and feoB was upregulated 4.11-fold and 2.71-fold in the presence of $640{\mu}M$ $Ga(NO_3)_3$, respectively. In conclusion, our study suggested that although $Ga(NO_3)_3$ could interfere with the growth of V. splendidus Vs, it could also stimulate both the production of $Fe^{3+}$-reducing HGA-melanin and the expression of feoA and feoB, which facilitate $Fe^{2+}$ transport in V. splendidus Vs.

Blue mold in citrus is caused by Penicillium italicum. In this study, the P. italicum-specific primers were developed for rapid detection based on the conserved genes RPB1 and RPB2 among Penicillium genomes. The two primer pairs RPB1-a and RPB1-b proved to be specific to detect P. italicum. The PCR assay among 39 fungal isolates and the colonial, pathogenic morphologies and molecular methods validated the specificity and reliability of these two primer pairs. This report provided a method and P. italicum-specific primers, which might greatly contribute to citrus postharvest industry.

Autophagy is crucial for immune defense against Mycobacterium tuberculosis (Mtb) infection. Mtb can evade host immune attack and survival within macrophages by manipulating the autophagic process. MicroRNAs (miRNAs) are small, non-coding RNAs that are involved in regulating vital genes during Mtb infection. The precise role of miRNAs in autophagy with the exits of Mtb remains largely unknown. In this study, we found miR-1958, a new miRNA that could regulate autophagy by interacting with 3'UTR of autophagy-related gene 5 (Atg5). In addition, Mtb infection triggered miR-1958 expression in RAW264.7 cells. What's more, miR-1958 overexpression blocked autophagic flux by impairing the fusion of autophagosomes and lysosomes. Overexpression of miR-1958 reduced Atg5 expression and LC3 puncta while inhibition of miR-1958 brought an increase of Atg5 and LC3 puncta; the opposite results were observed in detection of p62. The survival of Mtb in RAW264.7 cells transfected with mimic of miR-1958 was enhanced. Taken together, our research demonstrated that a novel miR-1958 could inhibit autophagy by interacting with Atg5 and favored intracellular Mtb survival in RAW264.7 cells.

Middle East respiratory syndrome coronavirus (MERS-CoV) was first identified in Saudi Arabia in 2012 and related infection cases have been reported in over 20 countries. Roughly 10,000 human cases have so far been reported in total with fatality rates at up to 40%. The majority of cases have occurred in Saudi Arabia with mostly sporadic outbreaks outside the country except for the one in South Korea in 2015. The Korean MERS-CoV strain was isolated from the second Korean patient and its genome was fully sequenced and deposited. To develop virus-specific protective and therapeutic agents against the Korean isolate and to investigate molecular determinants of virus-host interactions, it is of paramount importance to generate its full-length cDNA. Here we report that two full-length cDNAs from a Korean patient-isolated MERS-CoV strain were generated by a combination of conventional cloning techniques and efficient Gibson assembly reactions. The full-length cDNAs were validated by restriction analysis and their sequence was verified by Sanger method. The resulting cDNA was efficiently transcribed in vitro and the T7 promoter-driven expression was robust. The resulting reverse genetic system will add to the published list of MERS-CoV cDNAs and facilitate the development of Korean isolate-specific antiviral measures.