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The effects of grazed grass, grass silage, or concentrates on fatty acid composition and conjugated linoleic acid (cis-9, trans-11-18:2; CLA) concentrations of i.m. fat of steers fed to achieve similar carcass growth rates were investigated. Fifty steers were divided into 10 blocks based on body weight and assigned at random from within blocks to one of five dietary treatments. The experimental rations offered daily for 85 d preceding slaughter were 1) grass silage for ad libitum intake plus 4 kg of concentrate, 2) 8 kg of concentrate plus 1 kg of hay, 3) 6 kg of grazed grass DM plus 5 kg of concentrate, 4) 12 kg of grazed grass DM plus 2.5 kg concentrate, or 5) 22 kg of grazed grass DM. The concentration of polyunsaturated fatty acids (PUFA) in i.m. fat was higher (P < .05) for steers offered ration 5 than for those given any other ration. Decreasing the proportion of concentrate in the diet, which effectively increased grass intake, caused a linear decrease in the concentration of i.m. saturated fatty acids (SFA) (P < .01) and in the n-6:n-3 PUFA ratio (P < .001) and a linear increase in the PUFA:SFA ratio (P < .01) and the conjugated linoleic acid concentration (P < .001). The data indicate that i.m. fatty acid composition of beef can be improved from a human health perspective by inclusion of grass in the diet.

The macrocyclic polyketides rapamycin and FK506 are potent immunosuppressants that prevent T-cell proliferation through specific binding to intracellular protein receptors (immunophilins). The cloning and specific alteration of the biosynthetic genes for these polyketides might allow the biosynthesis of clinically valuable analogues. We report here that three clustered polyketide synthase genes responsible for rapamycin biosynthesis in Streptomyces hygroscopicus together encode 14 homologous sets of enzyme activities (modules), each catalyzing a specific round of chain elongation. An adjacent gene encodes a pipecolate-incorporating enzyme, which completes the macrocycle. The total of 70 constituent active sites makes this the most complex multienzyme system identiried so far. The DNA region sequenced (107.3 kbp) contains 24 additional open reading frames, some of which code for proteins governing other key steps in rapamycin biosynthesis.Polyketides are a large and highly diverse group of natural products that includes antibiotics, antitumor compounds, and immunosuppressants. The specific binding of polyketides to prevent T-cell proliferation was reported in 1992 by Schreiber (1) and Rosen and Schreiber (2). These polyketide metabolites are produced by successive condensation of simple carboxylic acid units (primarily acetate and propionate) as for fatty acid biosynthesis (3), except that the 3-keto function introduced during each elongation cycle may be reduced only partially or not at all. Macrocyclic polyketides are produced principally by Streptomyces and related filamentous bacteria, through the action of so-called type I modular polyketide synthases (PKSs), multienzymes in which different sets (modules) of enzymic activities catalyze each successive round of elongation, as first shown for the erythromycin-producing PKS (4-6). Characterization and genetic engineering of such systems to produce "hybrid" products (7) are particularly challenging because of the large size of the genes and their products and because the factors that control the specificity of chain extension are still largely unknown (7,8).Rapamycin ( Fig. 1) is a macrocyclic polyketide from Streptomyces hygroscopicus that, in addition to its antifungal (13) and antitumor (14) properties, is a potent immunosuppressant (15). Like the structurally related FK506, rapamycin is of interest for the clinical treatment of autoimmune disease (16) and in the prevention of rejection of organ and skin allografts (15,17). In spite of their similar polyketide backbone, these immunosuppressants act in radically different ways on T cells, FK506 by inhibiting the production of interleukin 2 (1, 2) and rapamycin by preventing the proliferative response to interleukin 2 bound at the interleukin 2 receptor (18). The engineered biosynthesis of altered rapamycins would also be of great interest for the study of these signaling processes. We have therefore undertaken a detailed study of the organization of the rapamycin biosynthetic genes in S. hygroscopicus....

Getting down to specifics: Key amino acid residues were found to correlate with ketoreductase domain stereospecificity in modular polyketide synthases. These residues may allow alcohol stereochemistry (see scheme; ACP, acyl carrier protein) in polyketides to be predicted from ketoreductase sequences. The results also suggest that polyketide synthase dehydratase domains have a preference for 3hydroxyacyl substrates with the same alcohol stereochemistry as the (3R)‐hydroxyacyl chains used by dehydratases in fatty acid synthases.

The availability of these genes and the development of a method for gene disruption and replacement in S. nodosus should allow production of novel amphotericins. A panel of analogues could lead to identification of derivatives with increased solubility, improved biological activity and reduced toxicity.

Modular polyketide synthases are multienzymes responsible for the biosynthesis of a large number of clinically important natural products. They contain multiple sets, or modules, of enzymatic activities, distributed between a few giant multienzymes and there is one module for every successive cycle of polyketide chain extension. We show here that each multienzyme in a typical modular polyketide synthase forms a (possibly helical) parallel dimer, and that each pair of identical modules interacts closely across the dimer interface. Such an arrangement would allow identical modules to share active sites for chain extension, and thus to function independently of flanking modules, which would have important implications both for mechanisms of evolution of polyketide synthases and for their future genetic engineering.

During assembly of complex polyketide antibiotics like erythromycin A, molecular recognition by the multienzyme polyketide synthase controls the stereochemical outcome as each successive methylmalonyl-coenzyme A (CoA) extender unit is added. Acylation of the purified erythromycin-producing polyketide synthase has shown that all six acyltransferase domains have identical stereospecificity for their normal substrate, (2S)-methylmalonyl-CoA. In contrast, the configuration of the methyl-branched centers in the product, that are derived from (2S)-methylmalonyl-CoA, is different. Stereoselection during the chain building process must, therefore, involve additional epimerization steps.

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