Biosynthesis of the immunosuppressants FK506, FK520, and rapamycin involves a previously undescribed family of enzymes acting on chorismate.

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 (4R,5R)-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 (4R,5R)-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 (4R,5R)-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.

An efficient stress-free strategy to displace stable bacterial plasmids.

A key stage in determining the phenotype(s) conferred by a plasmid is its displacement, or 'curing,' to create a plasmid-free strain. However, many plasmids are very stable, not only because they contain multiple replicons, but also because they can encode post-segregational killing systems that reduce the viability of plasmid-free segregants. We have developed an efficient curing strategy that involves combining key regions of the replicons and the post-segregational killing loci into an unstable cloning vector carrying sacB, which confers sensitivity to sucrose. Targeting plasmids of both the F family of Escherichia coli and the broad-host-range IncP-1 family, we demonstrated displacement of susceptible resident plasmids from all clones tested. Growth on sucrose allowed the isolation of many clones without either plasmid. This strategy is highly efficient and avoids the stress of inducing and surviving the effects of post-segregational killing systems or other lethal gene products.

Biosynthesis of the putative siderophore erythrochelin requires unprecedented crosstalk between separate nonribosomal peptide gene clusters.

The genome of the erythromycin-producing bacterium Saccharopolyspora erythraea contains many orphan secondary metabolite gene clusters including two (nrps3 and nrps5) predicted to govern biosynthesis of nonribosomal peptide-based siderophores. We report here the production by S. erythraea, even under iron-sufficient conditions, of a 2,5-diketopiperazine siderophore candidate we have named erythrochelin. Deletion of the nonribosomal peptide synthetase (NRPS) gene ercD within the nrps5 cluster abolished erythrochelin production. The tetrapeptide backbone of erythrochelin (alpha-N-acetyl-delta-N-acetyl-delta-N-hydroxyornithine-serine-delta-N-hydroxyornithine-delta-N-acetyl-delta-N-hydroxyornithine) suggests an orthodox colinear model for erythrochelin assembly. Curiously, the delta-N-acetyltransferase required for erythrochelin biosynthesis is encoded within a remote NRPS-cluster (nrps1) whose own NRPS contains an inactivating mutation. Disruption of the nrps1 gene mcd abolished erythrochelin biosynthesis, which could then be restored by addition of synthetic L-delta-N-acetyl-delta-N-hydroxyornithine, confirming an unprecedented example of functional crosstalk between nrps clusters.

Antigen-specific apheresis of pathogenic autoantibodies from myasthenia gravis sera.

Myasthenia gravis (MG) is usually caused by autoantibodies against muscle nicotinic acetylcholine receptor (AChR), which is composed of five subunits (alpha(2)betagammadelta or alpha(2)betaepsilondelta). Current treatments, including plasmapheresis, are nonspecific, causing several side effects. We aim to develop an antigen-specific alternative to plasmapheresis, since the latter removes indispensable plasma components in addition to anti-AChR antibodies. We are developing a method for the selective depletion of the anti-AChR autoantibodies from patients' plasma through the construction of "immunoadsorbent" columns carrying AChR domains. We have expressed the extracellular domains (ECDs, amino acids approximately 1-210/220) of all human muscle AChR subunits in Pichia pastoris and, in preliminary experiments, in E. coli. The ECDs were immobilized (individually or mixed) on Sepharose beads, producing Sepharose-ECD columns, which were tested for their immunoadsorbing capacity on MG sera and shown to specifically eliminate major autoantibody fractions from several MG sera. The immobilized ECDs remained stable and did not dissociate from their matrix after incubation with serum, whereas the procedure was neither toxic nor immunogenic in two experimental rabbits. Testing the intact or antibody-depleted MG sera and the affinity purified autoantibodies showed that both the intact sera and the purified autoantibodies, but not the antibody-depleted sera, could induce AChR loss in cell cultures and experimental MG in rats. This preliminary study suggests that the myasthenic potency of MG sera is entirely due to their anti-AChR antibodies and therefore their depletion should be of therapeutic value. We conclude that ECD-mediated immunoadsorption can be used as an efficient, antigen-specific therapy for MG.