The isolation of novel DEBS-derived octaketides provides the first evidence that an extension module in a modular PKS has the potential to catalyse iterative rounds of chain elongation like other type I FAS and PKS systems. The factors governing the extent of such 'stuttering' remain to be determined.

The strategy exemplified here shows far-reaching possibilities for combinatorial engineering of polyketide natural products, as well as revealing the ability of modular polyketide synthases to 'skip' extension modules. The results also provide additional insight into the three-dimensional arrangement of modules within these giant synthases.

These results demonstrate that, as predicted, even a single-module PKS is catalytically active in the absence of other DEBS proteins. In its normal context, the ketosynthase domain KS1 is thought to generate a (2S)-2methyl-3-hydroxy intermediate by epimerising the initial product of carbon-carbon chain formation, the (2R)-2-methyl-3-ketoester. The observed formation of the alternative (2R)-methyl-3-hydroxy product catalysed by DKS1-2 provides strong support for this proposal, and indicates how targeted alteration of stereospecificity can be achieved on a modular PKS.

In the photosynthetic bacteria Rhodospirillum rubrum and Rhodopseudomonas capsulatus posttranslational regulation of nitrogenase is due to ADP-ribosylation of the Fe-protein, the dinitrogenase reductase [Burris, R. H, (1991) J. Biol. Chem. 266,. This mechanism has been assumed to be responsible for nitrogenase modification in a variety of organisms. In the present study, we examined whether ADP-ribosylation holds true for the filamentous cyanobacterium Anabaena vuriabilis. Genes coding for the nitrogenase-modifying enzymes dinitrogenase reductase-activating glycohydrolase (DRAG) and dinitrogenase reductase ADP-ribosyl transferase (DRAT) from R. rubrum have been subcloned and overexpressed in Escherichia coli. After isolation under anaerobic conditions, both proteins were functional as determined by in-vitro assays using nitrogenase from R. rubrum as substrate. In contrast to the R. rubrum enzyme, nitrogenase from A. variabilis was not affected by DRAG or DRAT. Neither could inactive nitrogenase be restored by DRAG, nor nitrogenase activity suppressed by DRAT. Using specific antibodies against arginine-bound ADP-ribose The reduction of dinitrogen to ammonia during nitrogen fixation is catalyzed by a multi-enzyme complex consisting of dinitrogenase (MoFe-protein) and dinitrogenase reductase (usually referred to as Fe-protein; see [ l ] for a review). In the cyanobacterium Anabaena variabilis this enzyme complex is located in the heterocysts [2], cells with a thick, laminated envelope that functions as a diffusion barrier for gases. High respiration rates and lack of photosynthetic oxygen evolution enables these cells to fix nitrogen aerobically. Heterocysts do not catalyze CO, fixation and are dependent on adjacent photosynthetic vegetative cells for supply of fixed carbon. Since nitrogen fixation is an energy-demanding process (20-30 molecules ATP are required for the fixation of one molecule N2), regulation of nitrogenase requires both transcriptional and posttranslational events [ 11. Expression of nitrogenase in cyanobacteria depends on combined nitrogen, oxygen and cell differentiation [3, 41. In addition to transcriptional control, enzyme activity is influenced by external factors which limit the C supply of heterocysts 151 and by excess ammonium chloride at alkaline pH 161. These treatments cause inactivation of the enzyme ('ammonia switch-off'). In a number of diazotrophs, nitrogenase activity is regulated posttranslationally by reversible covalent ADP-ribosylation of the Fe-protein, as was most thoroughly investigated with the photosynthetic bacterium Rhodospirillum rubrum 17, 81. The site of ADP-ribosylation is a specific arginine residue (ArglOl or ArglOO in case of Azotobacter vinelandii) in one of the two identical subunits of the Fe-protein, thereby blocking an effective complex formation between the Fe-protein and . The process of addition and cleavage of the modifying group is catalyzed by specific enzymes. Modification by ADP-ribosylation (inactivation) is performed by dinitrogenase reductase AD...

A previously unidentified set of genes encoding a modular polyketide synthase (PKS) has been sequenced in Saccharopolyspora erythraea, producer of the antibiotic erythromycin. This new PKS gene cluster (pke) contains four adjacent large open reading frames (ORFs) encoding eight extension modules, flanked by a number of other ORFs which can be plausibly assigned roles in polyketide biosynthesis. Disruption of the pke PKS genes gave S. erythraea mutant JC2::pSBKS6, whose growth characteristics and pattern of secondary metabolite production did not apparently differ from the parent strain under any of the growth conditions tested. However, the pke PKS loading module and individual pke acyltransferase domains were shown to be active when used in engineered hybrid PKSs, making it highly likely that under appropriate conditions these biosynthetic genes are indeed expressed and active, and synthesize a novel polyketide product.