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....

Transitions towards sustainability are urgently needed to address the interconnected challenges of economic development, ecological integrity, and social justice, from local to global scales. Around the world, collaborative science-society initiatives are forming to conduct experiments in support of sustainability transitions. Such experiments, if carefully designed, provide significant learning opportunities for making progress on transition efforts. Yet, there is no broadly applicable evaluative scheme available to capture this critical information across a large number of cases, and to guide the design of transition experiments. To address this gap, the article develops such a scheme, in a tentative form, drawing on evaluative research and sustainability transitions scholarship, alongside insights from empirical cases. We critically discuss the scheme's key features of being generic, comprehensive, operational, and formative. Furthermore, we invite scholars and practitioners to apply, reflect and further develop the proposed tentative scheme -making evaluation and experiments objects of learning.

The amino acid sequences of a large number of polyketide synthase domains that catalyse the transacylation of either methylmalonyl-CoA or malonyl-CoA onto acyl carrier protein (ACP) have been compared. Regions were identified in which the acyltransferase sequences diverged according to whether they were specific for malonyl-CoA or methylmalonyl-CoA. These differences are sufficiently clear to allow unambiguous assignment of newly-sequenced acyltransferase domains in modular polyketide synthases. Comparison with the recently-determined structure of the malonyltransferase from Escherichia coli fatty acid synthase showed that the divergent region thus identified lies near the acyltransferase active site, though not close enough to make direct contact with bound substrate.

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