The antibiotic kijanimicin produced by the actinomycete Actinomadura kijaniata has a broad spectrum of bioactivities as well as a number of interesting biosynthetic features. To understand the molecular basis for its formation and to develop a combinatorial biosynthetic system for this class of compounds, a 107.6 kb segment of the Actinomadura kijaniata chromosome containing the kijanimicin biosynthetic locus was identified, cloned, and sequenced. The complete pathway for the formation of TDP-L-digitoxose, one of the two sugar donors used in construction of kijanimicin, was elucidated through biochemical analysis of four enzymes encoded in the gene cluster. Sequence analysis indicates that the aglycone kijanolide is formed by the combined action of a modular Type-I polyketide synthase (PKS) and a conserved operon involved attachment and intramolecular cyclization of a glycerate-derived three-carbon unit, which forms the core of the spirotetronate moiety. The genes involved in the biosynthesis of the unusual deoxysugar D-kijanose [2,3,4,6-tetradeoxy-4-(methylcarbamyl)-3-C-methyl-3-nitro-D-xylo-hexopyranose], including one encoding a flavoenzyme predicted to catalyze the formation of the nitro group, have also been identified. This work has implications for the biosynthesis of other spirotetronate antibiotics and nitro sugar-bearing natural products, as well as for future mechanistic and biosynthetic engineering efforts.Kijanimicin (1) is a spirotetronate antibiotic isolated from Actinomadura kijaniata, a soil actinomycete. It has a broad spectrum of antimicrobial activity against Gram-positive bacteria, anaerobes, and the malaria parasite Plasmodium falciparum, 1 and also shows antitumor activity. 2 The structure of kijanimicin (1) consists of a pentacyclic core, which is equipped with four L-digitoxose (2) units and a rare nitro sugar, 2,3,4,6-tetradeoxy-4-(methylcarbamyl)-3-C-methyl-3-nitro-D-xylo-hexopyranose, commonly known as D-kijanose (3). More than sixty kijanimicin-related spirotetronate-type compounds have been reported. Most are made by strains of high-GC Gram positive bacteria (Actinomycetes), including Streptomyces, 3-8 Micromonospora, 9-12 Actinomadura, 1,13,14 Saccharothrix, 15 and Verrucosispora. 16 A species of Bacillus has also been identified as a producer of a member of this class of compounds. 17 Nearly all members of this class exhibit both antibacterial and antitumor activities, and many possess other biological activities. Well-known examples include chlorothricins (4), the anticholesterolemic agents; 18,19 tetronothiodin, a cholecystokinin B (CCK-B) inhibitor; 4 MM46115, an antiviral drug effective against parainfluenzae virus 1 and 2; 13 and tetrocarcins (5) and arisostatins, both of which have been shown to have therapeutic potential as inducers of apoptosis. 20-23 In a recent study, a collection of tetrocarcin analogues was prepared synthetically and some of them showed improved apoptosis-inducing activity. 24 Hence, compounds of this class have broad therapeutic potential wort...

Microalgae have presented themselves as a strong candidate to replace diminishing oil reserves as a source of lipids for biofuels. Here we describe successful modifications of terrestrial plant lipid content which increase overall lipid production or shift the balance of lipid production towards lipid varieties more useful for biofuel production. Our discussion ranges from the biosynthetic pathways and rate limiting steps of triacylglycerol formation to enzymes required for the formation of triacylglycerol containing exotic lipids. Secondarily, we discuss techniques for genetic engineering and modification of various microalgae which can be combined with insights gained from research in higher plants to aid in the creation of production strains of microalgae.

Understanding the mode of action of small molecules is an integral facet of drug discovery. We report an optimized immunoaffinity fluorescent method that allows one to conduct parallel studies at both the cellular and molecular level using a single probe construct. Viability of the method has been evaluated analytically and applied using glycyrrhetic acid as a model.