Despite extensive similarities between the genomes of the Streptomyces temperate phages C31 and BT1, the attP-int loci are poorly conserved. Here we demonstrate that BT1 integrates into a different attachment site than C31. BT1 attB lies within SCO4848 encoding a 79-amino-acid putative integral membrane protein.Integration vectors based on BT1 integrase were shown to have a broad host range and are fully compatible with those based on the C31 attP-int locus.The attP-int locus from C31 has been heavily exploited in the construction of versatile, low-copy-number, and convenient vectors for use in a broad range of Streptomyces species (5, 10). Despite their wide use and clear advantages, it has been reported that integration of these vectors into the C31 attB site can cause detrimental effects on antibiotic production in some strains (2). C31 integrates intragenically into SCO3798, a highly conserved gene in prokaryotes and eukaryotes but is not essential for the growth of Streptomyces coelicolor in the laboratory (8). Although some phages can regenerate a functional gene after insertion (7), there is no evidence that this is the case with C31. Furthermore, a vector, pSET152 containing the C31 attP-int locus, introduced by conjugation from Escherichia coli can integrate into secondary or pseudo-attB sites in both S. coelicolor and Streptomyces lividans (8). The reported reductions in antibiotic synthesis could be caused by insertional mutagenesis into SCO3798 or by integration into one of the pseudo-attB sites or some other factor. Another potential problem with integrating vectors could be the absence of an efficiently recognized attB site in some streptomycete strains. Indeed, Saccharopolyspora erythraea appears to lack a C31 attB site (P. Leadlay, personal communication). For these reasons and as many workers would like to use two compatible integrating vectors in the same organism, we have investigated the integration site of the Streptomyces phage BT1, a homoimmune relative of C31. We demonstrate that BT1 does indeed integrate into a different attB site in S. coelicolor, and we have constructed novel integrating vectors derived from the BT1 attP-int locus.The organization of the BT1 genome is highly similar to that of C31, and the majority of gene products are closely related (9). There is evidence, however, of mosaicism between the two genomes where DNA has been inserted and/or deleted in one genome but not in the other, and there are sudden transitions in the level of sequence similarity (9). One of the most noticeable differences is the relatively poor sequence similarity of int and the three genes upstream, genes 26 to 28. BT1 integrase and gp26 to gp28 exhibit 26% and 10 to 18% identity to their C31 homologues, respectively. Despite this poor similarity, BT1 integrase is clearly a member of the large serine recombinase family, as it contains conserved motifs present in other members of this group (12). Furthermore, no significant similarity could be detected between the C31 attP site and any BT1 sequence....

Erythromycin, avermectin and rapamycin are clinically useful polyketide natural products produced on modular polyketide synthase multienzymes by an assembly-line process in which each module of enzymes in turn specifies attachment of a particular chemical unit. Although polyketide synthase encoding genes have been successfully engineered to produce novel analogues, the process can be relatively slow, inefficient, and frequently low-yielding. We now describe a method for rapidly recombining polyketide synthase gene clusters to replace, add or remove modules that, with high frequency, generates diverse and highly productive assembly lines. The method is exemplified in the rapamycin biosynthetic gene cluster where, in a single experiment, multiple strains were isolated producing new members of a rapamycin-related family of polyketides. The process mimics, but significantly accelerates, a plausible mechanism of natural evolution for modular polyketide synthases. Detailed sequence analysis of the recombinant genes provides unique insight into the design principles for constructing useful synthetic assembly-line multienzymes.

The macrocyclic polyketides FK506, FK520, and rapamycin are potent immunosuppressants that prevent T-cell proliferation through initial binding to the immunophilin FKBP12. Analogs of these molecules are of considerable interest as therapeutics in both metastatic and inflammatory disease. For these polyketides the starter unit for chain assembly is (4 R ,5 R )-4,5-dihydroxycyclohex-1-enecarboxylic acid derived from the shikimate pathway. We show here that the first committed step in its formation is hydrolysis of chorismate to form (4 R ,5 R )-4,5-dihydroxycyclohexa-1,5-dienecarboxylic acid. This chorismatase activity is encoded by fkbO in the FK506 and FK520 biosynthetic gene clusters, and by rapK in the rapamycin gene cluster of Streptomyces hygroscopicus . Purified recombinant FkbO (from FK520) efficiently catalyzed the chorismatase reaction in vitro, as judged by HPLC-MS and NMR analysis. Complementation using fkbO from either the FK506 or the FK520 gene cluster of a strain of S. hygroscopicus specifically deleted in rapK (BIOT-4010) restored rapamycin production, as did supplementation with (4 R ,5 R )-4,5-dihydroxycyclohexa-1,5-dienecarboxylic acid. Although BIOT-4010 produced no rapamycin, it did produce low levels of BC325, a rapamycin analog containing a 3-hydroxybenzoate starter unit. This led us to identify the rapK homolog hyg5 as encoding a chorismatase/3-hydroxybenzoate synthase. Similar enzymes in other bacteria include the product of the bra8 gene from the pathway to the terpenoid natural product brasilicardin. Expression of either hyg5 or bra8 in BIOT-4010 led to increased levels of BC325. Also, purified Hyg5 catalyzed the predicted conversion of chorismate into 3-hydroxybenzoate. FkbO, RapK, Hyg5, and Bra8 are thus founder members of a previously unrecognized family of enzymes acting on chorismate.

The fate of live forest biomass is largely controlled by growth and disturbance processes, both natural and anthropogenic. Thus, biomass monitoring strategies must characterize both the biomass of the forests at a given point in time and the dynamic processes that change it. Here, we describe and test an empirical monitoring system designed to meet those needs. Our system uses a mix of field data, statistical modeling, remotely-sensed time-series imagery, and small-footprint lidar data to build and evaluate maps of forest biomass. It ascribes biomass change to specific change agents, and attempts to capture the impact of uncertainty in methodology. We find that: • A common image framework for biomass estimation and for change detection allows for consistent comparison of both state and change processes controlling biomass dynamics. • Regional estimates of total biomass agree well with those from plot data alone.• The system tracks biomass densities up to 450-500 Mg ha −1 with little bias, but begins underestimating true biomass as densities increase further. • Scale considerations are important. Estimates at the 30 m grain size are noisy, but agreement at broad scales is good. Further investigation to determine the appropriate scales is underway. • Uncertainty from methodological choices is evident, but much smaller than uncertainty based on choice of allometric equation used to estimate biomass from tree data. • In this forest-dominated study area, growth and loss processes largely balance in most years, with loss processes dominated by human removal through harvest. In years with substantial fire activity, however, overall biomass loss greatly outpaces growth. Taken together, our methods represent a unique combination of elements foundational to an operational landscape-scale forest biomass monitoring program.