The biosynthetic gene cluster for lariatins A and B, anti-mycobacterial peptide antibiotics with a unique "lasso" structure, was cloned from Gram-positive bacterium Rhodococcus jostii K01-B0171. Random transposition mutagenesis using IS1415 derivative was carried out to identify a chromosomal locus involved in lariatin biosynthesis and six independent lariatin non-producing variants were obtained. Arbitrary PCR revealed that one insertion was located near the region involved in lariatin biosynthesis. Using the lariatin gene as a probe, a genomic library of R. jostii K01-B0171 was screened by colony hybridization, and two clones were obtained. Sequence analysis of these clones revealed that the gene cluster for lariatin biosynthesis spanning about 4.5 kb consisted of five open reading frames (larA to larE). We proposed that the linear precursor LarA is processed by LarB, LarC, and LarD, and the mature lariatin is exported by LarE.

Two isozymes for human acyl-coenzyme A:diacylglycerol acyltransferase (DGAT), DGAT1 and DGAT2, were independently expressed in DGAT-deficient Saccharomyces cerevisiae to establish DGAT1-and DGAT2-S. cerevisiae. The selectivity of DGAT inhibitors of natural origin towards the isozymes was assessed in enzyme assays using the microsomal fractions prepared from DGAT1-and DGAT2-S. cerevisiae. Amidepsines and xanthohumol inhibited DGAT1 and DGAT2 with similar potency, whereas roselipins were found to inhibit DGAT2 selectively. Keywords: acyl-CoA:diacylglycerol acyltransferase; amidepsine; DGAT; isozyme; roselipin; triacylglycerol; xanthohumol INTRODUCTION Triacylglycerol (TG) is the major energy-storage form of long-chain fatty acids in animals. 1,2 TG synthesis is important in many biological processes, including intestinal fat absorption, fat storage in adipocytes and energy metabolism in muscle, but excessive accumulation of TG in adipocytes as a result of a fat-rich diet or sedentary lifestyle causes obesity.Acyl-coenzyme A (CoA):diacylglycerol acyltransferase (DGAT, EC2.3.1.20) is a membrane-bound enzyme that catalyzes TG formation by acyl esterifications of diacylglycerol. Two biological pathways for TG synthesis, the glycerol phosphate pathway and the monoacylglycerol pathway, have been reported. These pathways form diacylglycerol (DG), which in turn is acylated by DGAT to form TG. 3 Recent molecular biological studies have revealed the existence of two different DGAT isozymes, DGAT1 and DGAT2, 4-6 in mammals, and extensive studies including biological experiments and knockout mice have shown that these isozymes have different functions in mammals. 7-13 Increased DGAT2 activity in the liver causes hepatic steatosis, whereas DGAT1 plays a role in very low-density lipoprotein (VLDL) synthesis in the liver and increases plasma VLDL concentration. Furthermore, newborn DGAT2-deficient mice die within hours of birth, whereas DGAT1-deficient mice are viable and have a modest reduction in tissue TG. Therefore, it is important to determine the selectivity of inhibitors towards the two DGAT isozymes for developing them as pharmaceutical drugs.Our research group conducted an enzyme assay involving rat liver microsomes to discover several DGAT inhibitors from natural sources,