Tyrosine is an important aromatic amino acid. Besides its nutritional value, tyrosine is also an important precursor for the synthesis of coumarins and flavonoids. Previously, our laboratory constructed a Saccharomyces cerevisiae strain LTH0 (ARO4K229L, ARO7G141S, Δaro10, Δzwf1, Δura3) where tyrosine feedback inhibition was released. In the present study, heterologous expression of betaxanthins synthesis genes DOD (from Mirabilis jalapa) and CYP76AD1 (from sugar beet B. vulgaris) in strain LTH0 enabled production of yellow fluorescence. The engineered strain LTH0-DOD-CYP76AD1 was subjected to UV combined with ARTP mutagenesis, followed by flow cytometry screening. Among the mutants screened, the fluorescence intensity of the mutant strain LTH2-5-DOD-CYP76AD1 at the excitation wavelength of 485 nm and emission wavelength of 505 nm was (5 941±435) AU/OD, which was 8.37 times higher than that of strain LTH0-DOD-CYP76AD1. Fourteen mutant strains were subjected to fermentation to evaluate their tyrosine producing ability. The highest extracellular tyrosine titer reached 26.8 mg/L, which was 3.96 times higher than that of strain LTH0-DOD-CYP76AD1. Heterologous expression of the tyrosine ammonia lyase FjTAL derived from Flavobacterium johnsoniae further increased the titer of coumaric acid to 119.8 mg/L, which was 1.02 times higher than that of the original strain LTH0-FjTAL.
Flavobacterium ; High-Throughput Screening Assays ; Mirabilis ; Saccharomyces cerevisiae/genetics* ; Tyrosine

Heparinase III is an enzyme that specifically cleaves certain sequences of heparan sulfate. Previous reports showed that this enzyme expressed in Escherichia coli was highly prone to aggregation in inclusion bodies and lacks detectable biological activity. In this paper, we fused a glutathione-S-transferase (GST) tag to the N-terminus of heparinase III gene and expressed the fusion protein in Escherichia coli to develop an expression system of soluble heparinase III. As a result, approximately 80% of the fusion protein was soluble. The protein was then purified to near homogeneity via one-step affinity chromatography. A 199.4-fold purification was achieved and the purified enzyme had a specific activity of 101.7 IU/mg protein. This represented 32.3% recovery of the total activity of recombinant GST-heparinase III. The maximum enzyme production was achieved when bacteria were induced with 0.5 mmol/L isopropyl-beta-D-thiogalactoside at 15 degrees C for 12 h. The enzyme showed maximum activity at 30 degrees C and pH 7.5. And the enzyme activity was stimulated by 1 mmol/L Ca2+ and 150 mmol/L NaCl.
Escherichia coli ; genetics ; metabolism ; Flavobacterium ; enzymology ; genetics ; growth & development ; Glutathione Transferase ; biosynthesis ; genetics ; Heparin Lyase ; biosynthesis ; genetics ; isolation & purification ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; isolation & purification

In order to obtain active recombinant PNGase F in Escherichia coli, a prokaryotic expression vector pET28a/PNGase F was constructed. Amplification of PNGase F was obtained using PCR technique employing suitable primers designed according to the PNGase F gene sequence from Flavobacterium nmeningosepticum. The expression of PNGase F gene in LB medium or M9 medium led to the formation of inclusion body and soluble protein, respectively. The refolding of the denatured inclusion body was successful by gradual dilution. Further purification of the refolded protein and soluble crude extract from M9 medium with Ni2+ -NTA argarose resulted a 90% purified PNGase F. The purified protein catalyzed the complete and intact cleavage of N-linked oligosaccharides from various glycoproteins. The efficiency of this cleavage was affected by the substrate status in the reaction system. In summary, we have developed an enzyme production system where PNGase F was over-expressed in recombinant E. coli. This system can provide more than 15 mg/L purified active PNGase F. This purified active PNGase F can be used as tools in analyzing the oligosaccharide structure.
Bacterial Proteins ; genetics ; metabolism ; Escherichia coli ; genetics ; metabolism ; Flavobacterium ; enzymology ; genetics ; Glycosylation ; Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase ; genetics ; metabolism ; Recombinant Fusion Proteins ; genetics ; metabolism

Bacterial cold water disease, enteric red mouth disease and frunculosis are the common bacterial diseases of fish worldwide. The etiologic agents of these diseases are Flavobacterium (F.) psychrophilum, Yersinia (Y.) ruckeri and Aeromonas (A.) salmonicida subsp. salmonicida, respectively. In this study, a multiplex polymerase chain reaction (m-PCR) method with YER8/10-Fer3/4-FP1/3 primer pairs which can identify these fish pathogens simultaneously was developed and optimized. In optimized conditions, neither false specific nor nonspecific amplification occurred. The detection limits of the m-PCR method using DNA extracts from dilutions of pure cultures of bacteria were 35 pg for Y. ruckeri and F. psychrophilum and 70 pg for A. salmonicida subsp. salmonicida. It was determined that 15 CFU Y. ruckeri and F. psychrophilum and 30 CFU A. salmonicida subsp. salmonicida could be detected by m-PCR developed using genomic DNA extracted from dilutions of the suspensions. The detection limits in the presence of tissue debris were 125 CFU for Y. ruckeri and F. psychrophilum and 250 CFU for A. salmonicida subsp. salmonicida. In conclusion, we submit that the m-PCR method developed and optimized in this study can be used for accurate and rapid identification of these bacteria.
Aeromonas salmonicida/*genetics ; Animals ; DNA Primers/genetics ; Fish Diseases/*diagnosis/*microbiology ; Fishes ; Flavobacterium/*genetics ; Gram-Negative Bacterial Infections/diagnosis/*veterinary ; Polymerase Chain Reaction/methods/*veterinary ; Yersinia rucker/*genetics

In order to evaluate the genetic variability of the causative agent of cold water disease (CWD), plasmid profiling was used to characterize Flavobacterium (F.) psychrophilum isolates (n = 169). Size analysis of plasmids in F. psychrophilum isolates (n = 128) from several fish species demonstrated that six kinds of plasmids were harbored, and ayu isolates had different profiles compared to other isolates. Moreover, multiple isolates (n = 41) from CWD outbreaks in 2002 to 2003 at a single ayu farm were examined to determine differences between isolates from successive outbreaks and showed different profiles by the sources of seedlings.
Animals ; DNA, Bacterial/genetics ; Disease Outbreaks/*veterinary ; Electrophoresis, Agar Gel/veterinary ; Fish Diseases/genetics/*microbiology ; Flavobacteriaceae Infections/microbiology/*veterinary ; Flavobacterium/genetics/*isolation & purification ; Genetic Variation/*genetics ; Japan ; *Osmeriformes ; Plasmids/genetics

A strain of Flavobacterium lindanitolerans isolated from a sick child's ascites was described. The 16S rRNA gene of the strain was 100% identical to that of Flavobacterium lindanitolerans which was first identified in India in 2008. It was first described that the isolate required X factor (Hemin) for growth in the optimal conditions of 37 °C with 5% CO(2). The isolate produced indole and H(2)S. It did not present hemolytic feature on blood agar.
Ascitic Fluid ; microbiology ; Child, Preschool ; Enterovirus A, Human ; isolation & purification ; Enterovirus Infections ; complications ; microbiology ; virology ; Fatal Outcome ; Flavobacteriaceae Infections ; complications ; microbiology ; virology ; Flavobacterium ; classification ; genetics ; isolation & purification ; Humans ; RNA, Ribosomal, 16S ; genetics ; Reverse Transcriptase Polymerase Chain Reaction

No abstract available.
Asian Continental Ancestry Group ; Flavobacteriaceae Infections ; Flavobacterium/*genetics/isolation & purification ; Humans ; Liver Cirrhosis, Alcoholic/blood/*diagnosis/microbiology ; Male ; Middle Aged ; RNA, Ribosomal, 16S/chemistry/genetics/metabolism ; Republic of Korea ; Sequence Analysis, DNA

No abstract available.
Aged ; Aneurysm/surgery ; Anti-Bacterial Agents/pharmacology ; Brain Diseases/surgery ; Craniotomy/adverse effects ; DNA, Bacterial/chemistry/genetics/metabolism ; Female ; Flavobacteriaceae Infections/etiology/microbiology ; Flavobacterium/classification/drug effects/*isolation & purification ; Humans ; Meningitis/*diagnosis/microbiology ; Microbial Sensitivity Tests ; Phylogeny ; Postoperative Complications ; Sequence Analysis, DNA