Role of the Phenylalanine-Hydroxylating System in Aromatic Substance Degradation and Lipid Metabolism in the Oleaginous Fungus Mortierella alpina

Mortierella alpina is a filamentous fungus commonly found in soil that is able to produce lipids in the form of triacylglycerols that account for up to 50% of its dry weight. Analysis of the M. alpina genome suggests that there is a phenylalanine-hydroxylating system for the catabolism of phenylalanine, which has never been found in fungi before. We characterized the phenylalanine-hydroxylating system in M. alpina to explore its role in phenylalanine metabolism and its relationship to lipid biosynthesis. Significant changes were found in the profile of fatty acids in M. alpina grown on medium containing an inhibitor of the phenylalanine-hydroxylating system compared to M. alpina grown on medium without inhibitor. Genes encoding enzymes involved in the phenylalanine-hydroxylating system (phenylalanine hydroxylase [PAH], pterin-4α-carbinolamine dehydratase, and dihydropteridine reductase) were expressed heterologously in Escherichia coli, and the resulting proteins were purified to homogeneity. Their enzymatic activity was investigated by high-performance liquid chromatography (HPLC) or visible (Vis)-UV spectroscopy. Two functional PAH enzymes were observed, encoded by distinct gene copies. A novel role for tetrahydrobiopterin in fungi as a cofactor for PAH, which is similar to its function in higher life forms, is suggested. This study establishes a novel scheme for the fungal degradation of an aromatic substance (phenylalanine) and suggests that the phenylalanine-hydroxylating system is functionally significant in lipid metabolism.     

Decreased expression of the DBC2 gene and its clinicopathological significance in breast cancer: correlation with aberrant DNA methylation

Loss of DBC2 (deleted in breast cancer 2) gene expression is frequent in breast cancer tissues. This can be explained by homozygous deletions or other mutations in a minority of cases but alternative mechanisms need to be investigated. Here, DBC2 expression was significantly suppressed compared with normal breast tissues in breast cancer tissues when analyzed by RT-PCR. Furthermore, DNA methylation on DBC2 was more prevalent in breast tumors than in normal tissues. DBC2 mRNA levels correlated with the degree of DBC2 methylation in breast cancer tissues and in a breast cancer cell line (T47D). Clinico-pathological correlation analysis showed that DBC2 promoter methylation was associated with tumor-node-metastasis stages II and III/IV, lymph node metastasis, p53 mutation, and HER2-positive status. Thus loss of DBC2 expression is caused by abnormal methylation of DBC2 and might have a role in breast cancer development.     

Metabolic engineering of Escherichia coli for enhanced biosynthesis of poly(3-hydroxybutyrate) based on proteome analysis

We have previously analyzed the proteome of recombinant Escherichia coli producing poly(3-hydroxybutyrate) [P(3HB)] and revealed that the expression level of several enzymes in central metabolism are proportional to the amount of P(3HB) accumulated in the cells. Based on these results, the amplification effects of triosephosphate isomerase (TpiA) and fructose-bisphosphate aldolase (FbaA) on P(3HB) synthesis were examined in recombinant E. coli W3110, XL1-Blue, and W lacI mutant strains using glucose, sucrose and xylose as carbon sources. Amplification of TpiA and FbaA significantly increased the P(3HB) contents and concentrations in the three E. coli strains. TpiA amplification in E. coli XL1-Blue lacI increased P(3HB) from 0.4 to 1.6 to g/l from glucose. Thus amplification of glycolytic pathway enzymes is a good strategy for efficient production of P(3HB) by allowing increased glycolytic pathway flux to make more acetyl-CoA available for P(3HB) biosynthesis.     

Expression of a lipase on the cell-surface of Escherichia coli using the OmpW anchoring motif and its application to enantioselective reactions

Microbial-surface display is the expression of proteins or peptides on the surface of cells by fusing an appropriate protein as an anchoring motif. Here, the outer membrane protein W (OmpW) was selected as a fusion partner for functional expression of Pseudomonas fluorescence SIK W1 lipase (TliA) on the cell-surface of Escherichia coli. Localization of the truncated OmpW-TliA fusion protein on the cell-surface was confirmed by immunoblotting and functional assay of lipase activity. Enantioselective hydrolysis of rac-phenylethyl butanoate by the displayed lipase resulted in optically active (R)-phenyl ethanol with 96 % enantiomeric excess and 44 % of conversion in 5 days. Thus, a small outer membrane protein OmpW, is a useful anchoring motif for displaying an active enzyme of ~50 kDa on the cell-surface and the surface-displayed lipase can be employed as an enantioselective biocatalyst in organic synthesis.     

Myosin-cross-reactive antigens from four different lactic acid bacteria are fatty acid hydratases

The 67 kDa myosin-cross-reactive antigen (MCRA) is a member of the MCRA family of proteins present in a wide range of bacteria and was predicted to have fatty acid isomerase function. We have now characterised the catalytic activity of MCRAs from four LAB stains, including Lactobacillus rhamnosus LGG, L. plantarum ST-III, L. acidophilus NCFM and Bifidobacterium animalis subsp. lactis BB-12. MCRA genes from these strains were cloned and expressed in Escherichia coli, and the recombinant protein function was analysed with lipid profiles by GC–MS. The four MCRAs catalysed the conversion of linoleic acid and oleic acid to their respective 10-hydroxy derivatives, which suggests that MCRA proteins catalyse the first step in conjugated linoleic acid production. This is the first report of MCRA from L. rhamnosus with such catalytic function.     

Maritime Legislation of Mainland China and Taiwan: Developments,Comparison, Implications,and Potential Challenges for the United States

In January 1998, Taiwan promulgated the Law of the Republic of China (ROC) on the Territorial Sea and the Contiguous Zone and the Law of the Republic of China on the Exclusive Economic Zone (EEZ) and the Continental Shelf, both of which came into force on January 21, 1998. On June 26, 1998, China adopted the Law of the People's Republic of China on the Exclusive Economic Zone and the Continental Shelf, which is the most significant maritime legislation since its 1992 Law on the Territorial Sea and the Contiguous Zone. This article studies the recent maritime legislation developments in Mainland China and Taiwan, the differences and similarities between the two sets of laws, their implications for the Asia-Pacific region, and the potential challenges for the United States.     

The Potential Marine Pollution Threat from Oil and Gas Development Activities in the Disputed South China Sea/Spratly Area: A Role that Taiwan Can Play

This article examines the potential threat of marine pollution caused by offshore oil and gas development activities in the disputed areas of the South China Sea (SCS) and the Spratly Islands. After addressing the potential threat of marine pollution, it discusses the legal obligations and political commitment of the SCS littoral states regarding the protection of the marine environment in the area. The role that Taiwan can play in these matters is also examined.     

Isolation of chicken embryonic stem cell and preparation of chicken chimeric model

Chicken embryonic stem cells (ESCs) were separated from blastoderms at stage-X and cultured in vitro. Alkaline phosphatase activity and stage-specific embryonic antigen-1 staining was conducted to detect ESCs. Then, chicken ESCs were transfected with linearized plasmid pEGFP-N1 in order to produce chimeric chicken. Firstly, the optimal electrotransfection condition was compared; the results showed the highest transfection efficiency was obtained when the field strength and pulse duration was 280 V and 75 μs, respectively. Secondly, the hatchability of shedding methods, drilling a window at the blunt end of egg and drilling a window at the lateral shell of egg was compared, the results showed that the hatchability was the highest for drilling a window at the lateral shell of egg. Thirdly, the hatchability of microinjection (ESCs was microinjected into chick embryo cavity) was compared too, the results showed there were significant difference between the injection group transfected with ESCs and that of other two groups. In addition, five chimeric chickens were obtained in this study and EGFP gene was expressed in some organs, but only two chimeric chicken expressed EGFP gene in the gonad, indicating that the chimeric chicken could be obtained through chick embryo cavity injection by drilling a window at the lateral shell of egg.