Biosynthesis of fatty acid ethyl ester (FAEE) by genetically engineered Escherichia coli has attracted extensive attentions from scientific community. In this study, we evaluated the effects of thioesterase with different origins on FAEE production and the results show that Cc FatB1 from Cinnamomum camphorum is better than tesA' from E. coli for FAEE production. Then, the optimized FAEE-producing strain KC4, with 21.4 mg/(L x OD600) FAEE production under flask condition and 31.16 mg/(L x OD600) under 5 L fermentation condition, was constructed by co-expression of Cc FatB1 and tesA'. Compared with the reported FAEE-producing strain KC3, KC4 possesses the higher FAEE productivity.
Escherichia coli ; genetics ; metabolism ; Esters ; metabolism ; Ethanol ; metabolism ; Fatty Acids ; biosynthesis ; Fermentation ; Genetic Engineering ; Thiolester Hydrolases ; genetics ; metabolism

To study the roles of glucosylglycerol phosphate synthase (Ggps) in glucosylglycerol (GG) and glycerol biosynthesis, we over-expressed Ggps from either Synechocystis sp. PCC 6803 or Synechococcus sp. PCC 7002 in a Synechocystis strain with a high GG titer, and determined the GG and glycerol accumulation in the resultant mutants grown under different NaCl-stress conditions. Ion chromatography results revealed that GG yield was not improved, but glycerol production was significantly enhanced by over-expression of Ggps from Synechocystis sp. PCC 6803 (6803ggpS). In addition, increasing the NaCl concentration of medium from 600 to 900 mmol/L led to a further 75% increase of glycerol accumulation in the mutant strain with 6803ggpS over-expression. These findings show the role of ggpS in driving the carbon flux to the glycerol biosynthesis pathway, and will be helpful for further improvement of GG and glycerol production in Synechocystis.
Bacterial Proteins ; metabolism ; Culture Media ; Glucosides ; biosynthesis ; Glucosyltransferases ; metabolism ; Glycerol ; metabolism ; Industrial Microbiology ; Sodium Chloride ; Synechococcus ; enzymology ; Synechocystis ; enzymology ; metabolism

Objective To identify the species of brucella rapidly with small fragments of PCR products. Methods The primers were designed according to Brucella spp. repeated insertion sequence IS711 in addition to the specific gene sequences of Brucella melitensis, Brucella abortus and Brucella suis. The small fragments of 3 standard strains and 13 clinical isolates of Brucella were amplified by PCR. Then PCR products were T-A cloned and sequenced. The DNA of standard strains were diluted for sensitivity and stability testing. Results Four specific PCR products were obtained by PCR (63, 67, 81 and 83 bp). The results of T-A cloning and sequencing were in accord with the target gene fragment test. The detection limit of DNA was 1 μg/L. The expected results were achieved with different primers. Conclusion Species identification of Brucella with small fragments of PCR production is a method to identify Brucella strains with great specificity,sensitivity and stability.

Objective To investigate the value of timely duodenal papilla fenestration for reducing the incidence of pancreatitis after endoscopic retrograde cholangiopancreatography (ERCP).Methods The clinical data of 181 patients with difficult biliary cannulatian during ERCP at the Xinhua Hospital of Shanghai Jiaotong University from July 2006 to December 2009 were retrospectively analyzed.Of the 181 patients,98 patients who received traditional incubation were in the control group,and the other 83 patients who received early duodenal papilla fenestration were in the test group.The success rate of selective incubation and incidence of pancreatitis were compared between the 2 groups.All data were analyzed using the t test,chi-square test or Wilcoxon rank sum test.Results The success rate of incubation,incidences of hyperamylasemia and pancreatitis were 85.7％ ( 84/98 ),7.1％ (7/98) and 10.2％ ( 10/98 ) in the control group,and 94.0％ ( 78/83 ),18.1％ ( 15/83 ) and 2.4％ (2/83) in the test group,respectively,and there were significant differences between the 2 groups (x2 =10.12,5.03,4.41,P＜0.05).The numbers of patients with mild,moderate and severe pancreatitis were 3,5 and 2 in the control group,and 1,1,0 in the test group,respectively,and there was a significant difference between the 2 groups ( Z =- 2.11,P ＜ 0.05 ).Conclusion Timely duodenal papilla fenestration is safe and effective in reducing the incidence of pancreatitis for patients with difficult biliary cannulation during ERCP.

AIM: To explore the molecular mechanism of asthenospermia(AST) by preliminary screening of nucleotide sequences from the ND3 and ND4L genes of mitochondrial DNA(mtDNA). METHODS: Samples from 50 AST patients and 42 age-matched normal controls were collected according to the WHO criteria. Density gradient centrifugation was applied to separate spermatozoa with different vigor. The ND3 and ND4L genes of mtDNA were amplified and sequenced directly from the extracted genomic DNA from AST patients and normal controls. The sequences were compared with revised Cambridge Reference Sequence(rCRS) to analyze the variants. RESULTS: A total of 22 nucleotide variations were found in ND3 and ND4L genes of mtDNA in asthenospermia group and control group. G10320A, A10398G and T10609C were missense mutations, while A10157G and A10313C were the reported for the first time in this study. Haplotype N in patients with AST(33/50) was higher than that in control group(14/42, P<0.05), and haplotype R9 in patients with AST(15/50) was also higher than that in control group(4/42, P<0.05) through genetic testing of ND3 gene. Rates of sperm progressive motility of haplotype F1, F2 and R9 were significantly lower than those of haplotype M and M rest. Two haplotype differences, haplotype M and N, were found in the same AST patient's spermatozoas which had different vigor. Haplotype M had stronger vigor, while haplotype N had lower vigor. By sequencing ND3 gene of mtDNA from 50 AST patients, we detected G10310A heteroplasmic mutation in 2 specimens of asthenospermia with poor and moderate motility spermatozoa, respectively. No mutation occurred in good motility spermatozoa. CONCLUSION: Haplotype of mitochondrial may have some correlation with sperm motility. The nt10398G-10400T polymorphisms may have benefit for sperm motility, whereas the mutation in nt10310A may impair sperm motility.

To evaluate the efficacy of HbA1C in diagnosing diabetes in subjects with primary hypertension.The results demonstrated that the area under the reciever operating characteristic ( ROC ) curve was 0.895,with corresponding sensitivity of 85.4％,and specificity of 82.2％,when the optimal cutpoint of HbA1C in diagnosing diabetes was 6.0％.Our study suggested that HbA1C ≥6.0％ can be used efficiently in diagnosing diabetes in patients with primary hypertension.

Free fatty acid profiles of wild type and fatty acyl-ACP synthase deletion mutant strain of Synechocystis sp. PCC6803 indicated that one origin of these fatty acids is the process of lipid remodeling or lipid degradation. Lipase is the key enzyme involved in this process. The gene sll1969 is the sole gene encodes a putative lipase in Synechocystis sp. PCC6803. To identify the function of this gene and its role in fatty acid metabolism, we cloned the sll1969 from genomic DNA, overexpressed it in Escherichia coli BL21 (DE3) using pET expression system and purified this recombinant enzyme with Nickel-nitrilotriacetic acid affinity chromatography. The enzyme activity was assayed by spectrophotometric with p-nitro-phenylbutyrate as substrate. The K(m) and k(cat) of the enzyme is (1.16 +/- 0.01) mmol/L and (332.8 +/- 10.0)/min, respectively toward p-nitro-phenylbutyrate at 30 degrees C. The optimal temperature of the enzyme is 55 degrees C. To investigate the biological role of Sll1969 in fatty acid metabolism in cyanobacteria, we constructed sll1969 deletion and overexpression mutant strains in the background of fatty acyl-ACP synthase deletion mutant of Synechocystis sp. PCC6803. The analyses of the content of free fatty acids in different mutant strains showed that the contents of Sll1969 and free fatty acid are positively correlated. The free fatty acid profiles of the sll1969 mutant strains suggested this enzyme is not the sole enzyme for degrading lipid in Synechocystis sp. PCC6803.
Escherichia coli ; genetics ; metabolism ; Fatty Acids, Nonesterified ; metabolism ; Lipase ; biosynthesis ; genetics ; Membrane Lipids ; genetics ; metabolism ; Mutation ; Recombinant Proteins ; biosynthesis ; genetics ; metabolism ; Synechocystis ; enzymology ; genetics ; metabolism

For metabolic engineering of cyanobacteria, there is an urgent need to construct a group of efficient heterologous gene expression platforms and to evaluate their expression efficiencies. Here we constructed three integrative vectors, the pKW1188-derived pFQ9F, pFQ9R and pFQ20, for integration of heterologous genes into the genome of the model cyanobacteria strain Synechocystis sp. strain PCC6803. The pFQ16, an RSF1010-derived broad host range shuttle vector, was constructed for conjugative transfer of genes to various cyanobacteria strains. All the four platforms constructed here applied the rbc (encodes Ribulose-1, 5-bisphosphate carboxylase/oxygenase) and the rbc terminator to promote and terminate the gene transcription. Besides, a "Shine-Dalgarno -AUG" fusion translation strategy was used to keep the high protein translation efficiency. Using lacZ as a reporter gene, the expression efficiency of pFQ20 was evaluated and showed a strong beta-galactosidase expression (109 Miller). Furthermore, the platform pFQ20 was used to express the E. coli tesA' gene and showed significant protein bands through the Western Blot test. The expression platforms constructed in this study offer useful molecular tools for metabolic engineering of cyanobacteria in the future.
Genetic Vectors ; genetics ; Industrial Microbiology ; methods ; Metabolic Engineering ; methods ; Palmitoyl-CoA Hydrolase ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; Synechocystis ; genetics ; metabolism ; beta-Galactosidase ; biosynthesis ; genetics

Cyanobacteria are a phylum of bacteria which are believed to be the oldest photosynthetic prokaryotic microorganisms on earth. The phylogenetic group of cyanobacteria was thought to be one of the prokaryotes that contain monoploid, oligoploid and polyploid species, and one obstacle to engineering cyanobacteria is their polyploidy genome. In recent years, the ploidy level of cyanobacteria was found to be influenced by growth phase and by multiple genetic and environmental factors. In the present article, we reviewed the progress, analytical methods and influencing factors on the cyanobacterial ploidy, and discussed the significance of cyanobacterial polyploidy regarding to environmental ecology and biotechnology. Based on this observation, the future research directions in this field are prospected.

ω-transaminases are able to catalyze the reversible transfer of amino groups between diverse amino compounds (such as amino acids, alkyl amines, aromatic amines) and carbonyl compounds (such as aldehydes, ketones, ketoacids). ω-transaminases exhibit great application prospects in the field of chiral amine biosynthesis because of their desirable properties, such as wide range of substrates, high stereoselectivity, and mild catalytic conditions. It is therefore important for China to develop efficient, specific, and environment-friendly chiral amine production technologies with independent intellectual property rights, which is of great significance for the development of pharmaceutical, pesticide, and material industries. This review systematically summarizes the Chinese patents regarding ω-transaminase filed by Chinese institutions in the recent decade. The development of ω-transaminase resource, enzymatic property improvement by protein engineering, application in chiral amine synthesis, and development of production technologies are elaborated. This review will shed light on further basic and application studies of ω-transaminase.
Transaminases/genetics* ; Amino Acids ; China ; Aldehydes ; Amines