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....

The ascomycetous fungus Nectria haematococca, (asexual name Fusarium solani), is a member of a group of >50 species known as the “Fusarium solani species complex”. Members of this complex have diverse biological properties including the ability to cause disease on >100 genera of plants and opportunistic infections in humans. The current research analyzed the most extensively studied member of this complex, N. haematococca mating population VI (MPVI). Several genes controlling the ability of individual isolates of this species to colonize specific habitats are located on supernumerary chromosomes. Optical mapping revealed that the sequenced isolate has 17 chromosomes ranging from 530 kb to 6.52 Mb and that the physical size of the genome, 54.43 Mb, and the number of predicted genes, 15,707, are among the largest reported for ascomycetes. Two classes of genes have contributed to gene expansion: specific genes that are not found in other fungi including its closest sequenced relative, Fusarium graminearum; and genes that commonly occur as single copies in other fungi but are present as multiple copies in N. haematococca MPVI. Some of these additional genes appear to have resulted from gene duplication events, while others may have been acquired through horizontal gene transfer. The supernumerary nature of three chromosomes, 14, 15, and 17, was confirmed by their absence in pulsed field gel electrophoresis experiments of some isolates and by demonstrating that these isolates lacked chromosome-specific sequences found on the ends of these chromosomes. These supernumerary chromosomes contain more repeat sequences, are enriched in unique and duplicated genes, and have a lower G+C content in comparison to the other chromosomes. Although the origin(s) of the extra genes and the supernumerary chromosomes is not known, the gene expansion and its large genome size are consistent with this species' diverse range of habitats. Furthermore, the presence of unique genes on supernumerary chromosomes might account for individual isolates having different environmental niches.

Each major step leading to the classical yellow, orange and red constituents of Monascus azaphilone pigments was defined.

The overall architecture of the gene cluster responsible for epothilone biosynthesis has been determined. The availability of the cluster should facilitate the generation of designer epothilones by combinatorial biosynthesis approaches, and the heterologous expression of epothilones in surrogate microbial hosts.