Heterologous Biosynthesis of the Sterol O-Acyltransferase Inhibitor Helvamide Unveils an α-Ketoglutarate-Dependent Cross-Linking Oxygenase.

We have identified the biosynthetic gene cluster (hvm) for the sterol O-acyltransferase inhibitor helvamide (1) from the genome of Aspergillus rugulosus MST-FP2007. Heterologous expression of hvm in A. nidulans produced a previously unreported analog helvamide B (5). An α-ketoglutarate-dependent oxygenase Hvm1 was shown to catalyze intramolecular cyclization of 1 to yield 5. The biosynthetic branch to the related hancockiamides and helvamides was found to be controlled by the substrate selectivity of monomodular nonribosomal peptide synthetases.

Fusion Enzymes Involved in Biosynthetic Tailoring Reactions in Fungi.

Enzyme fusion, the fusion of enzymes with different domains to a single protein, has been widely recognized as a promising strategy in the development of biocatalysts. Nature has evolved gene fusion to combine different catalytic enzymes to function as a fusion enzyme, and this strategy is utilized in many natural product biosynthetic pathways. Owing to rapid advances in genome sequencing and biosynthetic pathway characterization, there is increasing interest in fusion enzymes from fungal biosynthetic pathways, particularly those involved in tailoring steps. This concept aims to provide an up-to-date overview of fusion enzymes that catalyze tailoring reactions in the biosynthesis of fungal secondary metabolites. Since fungal fusion enzymes are often associated with novel metabolites, this pioneering work may stimulate the exploration of the structural diversity of fungal natural products through genome mining of the untapped biosynthetic pathways involving fusion enzymes.

Cloning of the non-structural gene 3 of hepatitis C virus and its inducible expression in cultured cells.

AIM:To study the inducible expression of hepatitis C virus ns3 gene (HCV ns3) in eukaryotic cells.METHODS: The ns3 gene was obtained from plasmid pBns3 by polymerase chain reaction and inserted into the cloning vector pGEM-T. Then, the ns3 was subcloned into the vector pMSG to generate dexamethasone (DM)-inducible expression plasmid pMSG-ns3.CHO cells were transfected by pMSG-ns3 using calcium phosphate precip-itation method and cultivated for 12h-24h. The transfected cells were induced with DM and the transient expression of NS3 protein was analyzed by ELISA and Western-blot methods.RESULTS:After treated with 3X10(-8)mol/L DM, the expression of NS3 was observed in the transfected CHO cells. A slightly higher level of NS3 was shown along with the time of DM treatment.CONCLUSION:The inducible expressing vector pMSG-ns3 might be helpful for further studies of the characteristics of the ns3 gene in vivo.