Genetically engineered analogs of ascomycin for nerve regeneration.

The polyketides FK506 (tacrolimus) and FK520 (ascomycin) are potent immunosuppressants that function by inhibiting calcineurin phosphatase through formation of an FKBP12-FK506/520-calcineurin ternary complex. They also have calcineurin-independent neuroregenerative properties in cell culture and animal models of nervous system disorders. Based on the crystal structure of the FKBP12-FK506-calcineurin complex, we deduced that the 13- and 15-methoxy groups of FK506 or FK520 are important for inhibition of calcineurin phosphatase but not for binding to FKBP12. By genetic modification of the FK520 gene cluster, we generated 13- and 15-desmethoxy analogs of FK520 that contain hydrogen, methyl, or ethyl instead of methoxy at one or both of these positions. These analogs bind FKBP12 tightly, have decreased calcineurin phosphatase inhibition and immunosuppressive properties, and enhance neurite outgrowth in cell cultures. A representative compound was also shown to accelerate nerve regeneration and functional recovery in the rat sciatic nerve crush model.

Beta-ketoacyl acyl carrier protein synthase III (FabH) is essential for fatty acid biosynthesis in Streptomyces coelicolor A3(2).

The Streptomyces coelicolor fab (fatty acid biosynthesis) gene cluster (fabD-fabH-acpP-fabF) is cotranscribed to produce a leaderless mRNA transcript. One of these genes, fabH, encodes a ketoacyl synthase III that is essential to and is proposed to be responsible for initiation of fatty acid biosynthesis in S. coelicolor.

The FK520 gene cluster of Streptomyces hygroscopicus var. ascomyceticus (ATCC 14891) contains genes for biosynthesis of unusual polyketide extender units.

FK520 (ascomycin) is a macrolide produced by Streptomyces hygroscopicus var. ascomyceticus (ATCC 14891) that has immunosuppressive, neurotrophic and antifungal activities. To further elucidate the biosynthesis of this and related macrolides, we cloned and sequenced an 80kb region encompassing the FK520 gene cluster. Genes encoding the three polyketide synthase (PKS) subunits (fkbB, fkbC and fkbA), the peptide synthetase (fkbP), the 31-O-methyltransferase (fkbM), the C-9 hydroxylase (fkbD) and the 9-hydroxyl oxidase (fkbO) had the same organization as the genes reported in the FK506 gene cluster of Streptomyces sp. MA6548 (Motamedi, H., Shafiee, A., 1998. The biosynthetic gene cluster for the macrolactone ring of the immunosuppressant FK506. Eur. J. Biochem. 256, 528-534). Disruption of a PKS gene in the cluster using the φC31 phage vector, KC515, led to antibiotic non-producing strains, proving the identity of the cluster. Previous labeling data have indicated that FK520 biosynthesis uses novel polyketide extender units (Byrne, K.M., Shafiee, A., Nielson, J., Arison, B., Monaghan, R.L., Kaplan, L., 1993. The biosynthesis and enzymology of an immunosuppressant, immunomycin, produced by Streptomyces hygroscopicus var, ascomyceticus. Dev. Ind. Microbiol. 32, 29-45). Genes in the flanking regions of the FK520 cluster were identified that appear to be involved in synthesis of these extender units. All but two of these genes were homologous to genes with known function. In addition to a crotonyl-CoA reductase gene (fkbS), at least two other genes are proposed to be involved in biosynthesis of the atypical PKS extender unit ethylmalonyl-CoA, which accounts for the ethyl side chain on C-21 of FK520. A set of five contiguous genes (fkbGHIJK) is proposed to be involved in biosynthesis of an unusual PKS extender unit bearing an oxygen on the alpha-carbon, and leading to the 13- and 15-methoxy side chains. These putative precursor synthesis genes in the flanking regions of the FK520 cluster are not found in the flanking regions of the rapamycin cluster (Molnár, I., Aparicio, J.F., Haydock, S.F., Khaw, L.E., Schwecke, T., König, A., Staunton, J., Leadlay, P.F., 1996. Organisation of the biosynthetic gene cluster for rapamycin in Streptomyces hygroscopicus: analysis of genes flanking the polyketide synthase. Gene 169, 1-7), consistent with labeling data showing that rapamycin biosynthesis uses only malonyl and methylmalonyl extender units.

The granaticin biosynthetic gene cluster of Streptomyces violaceoruber Tü22: sequence analysis and expression in a heterologous host.

BACKGROUND: The granaticins are members of the benzoisochromanequinone class of aromatic polyketides, the best known member of which is actinorhodin made by Streptomyces coelicolor A3(2). Genetic analysis of this class of compounds has played a major role in the development of hypotheses about the way in which aromatic polyketide synthases (PKSs) control product structure. Although the granaticin nascent polyketide is identical to that of actinorhodin, post-PKS steps involve different pyran-ring stereochemistry and glycosylation. Comparison of the complete gene clusters for the two metabolites is therefore of great interest. RESULTS: The entire granaticin gene cluster (the gra cluster) from Streptomyces violaceoruber T-22 was cloned on either of two overlapping cosmids and expressed in the heterologous host, Streptomyces coelicolor A3(2), strain CH999. Chemical analysis of the recombinant strains demonstrated production of granaticin, granaticin B, dihydrogranaticin and dihydrogranaticin B, which are the four known metabolites of S. violaceoruber. Analysis of the complete 39,250 base pair sequence of the insert of one of the cosmids, pOJ466-22-24, revealed 37 complete open reading frames (ORFs), 15 of which resemble ORFs from the act (actinorhodin) gene cluster of S. coelicolor A3(2). Among the rest, nine resemble ORFs potentially involved in deoxysugar metabolism from Streptomyces spp. and other bacteria, and six resemble regulatory ORFs. CONCLUSIONS: On the basis of these resemblances, putative functional assignments of the products of most of the newly discovered ORFs were made, including those of genes involved in the PKS and tailoring steps in the biosynthesis of the granaticin aglycone, steps in the deoxy sugar pathway, and putative regulatory and export functions.

Heterologously expressed acyl carrier protein domain of rat fatty acid synthase functions in Escherichia coli fatty acid synthase and Streptomyces coelicolor polyketide synthase systems.

INTRODUCTION: Fatty acid synthases (FASs) catalyze the de novo biosynthesis of long-chain saturated fatty acids by a process common to eubacteria and eukaryotes, using either a set of monofunctional proteins (Type II FAS) or a polypeptide containing several catalytic functions (Type I FAS). To compare the features of a Type I domain with its Type II counterpart we expressed and characterized an acyl carrier protein (ACP) domain of the Type I rat FAS. RESULTS: An ACP domain of rat FAS was defined that allows expression of a small percentage of active holo-ACP both in Escherichia coli, increasing fivefold upon co-expression with an E. coli holo-ACP synthase, and in Streptomyces coelicolor. The rat ACP domain functions with some components of the E. coli FAS, and can replace the actinorhodin polyketide synthase (PKS) ACP in S. coelicolorA3(2). Purification of the rat ACP domain from E. coli resulted in loss of its functionality. Purified apo-ACP could be converted to its holo-form upon incubation with purified E. coli holo-ACP synthase in vitro, however, suggesting that the loss of functionality was not due to a conformational change. CONCLUSIONS: Functionality of the recombinant rat ACP was shown in distantly related and diverse enzyme systems, suggesting that Type I and Type II ACPs have a similar conformation. A procedure was described that might permit the production of rat FAS holo-ACP for structural and further biochemical characterization.

Relationships between fatty acid and polyketide synthases from Streptomyces coelicolor A3(2): characterization of the fatty acid synthase acyl carrier protein.

We have characterized an acyl carrier protein (ACP) presumed to be involved in the synthesis of fatty acids in Streptomyces coelicolor A3(2). This is the third ACP to have been identified in S. coelicolor; the two previously characterized ACPs are involved in the synthesis of two aromatic polyketides: the blue-pigmented antibiotic actinorhodin and a grey pigment associated with the spore walls. The three ACPs are clearly related. The presumed fatty acid synthase (FAS) ACP was partially purified, and the N-terminal amino acid sequence was obtained. The corresponding gene (acpP) was cloned and sequenced and found to lie within 1 kb of a previously characterized gene (fabD) encoding another subunit of the S. coelicolor FAS, malonyl coenzyme A:ACP acyl-transferase. Expression of S. coelicolor acpP in Escherichia coli yielded several different forms, whose masses corresponded to the active (holo) form of the protein carrying various acyl substituents. To test the mechanisms that normally prevent the FAS ACP from substituting for the actinorhodin ACP, acpP was cloned in place of actI-open reading frame 3 (encoding the actinorhodin ACP) to allow coexpression of acpP with the act polyketide synthase (PKS) genes. Pigmented polyketide production was observed, but only at a small fraction of its former level. This suggests that the FAS and PKS ACPs may be biochemically incompatible and that this could prevent functional complementation between the FAS and PKSs that potentially coexist within the same cells.

Polyketide synthase acyl carrier proteins from Streptomyces: expression in Escherichia coli, purification and partial characterisation.

Acyl carrier proteins (ACPs) of the type II polyketide synthases for the aromatic antibiotics actinorhodin, granaticin, frenolicin and oxytetracycline were expressed in Escherichia coli downstream of an inducible phage T7 promoter. For the act and otc genes, several of the first eight codons were changed to synonymous codons used in highly expressed E. coli genes. Correlated with these changes, the amounts of the act and otc ACPs purified from the recombinant E. coli cultures were an order of magnitude greater than for the gra and fren ACPs expressed from the unmodified genes. Electrospray mass spectrometry (ESMS) of the purified proteins confirmed their calculated M(r) based on the DNA sequences while also revealing that, in the act and gra ACP samples, some 2% and 30% of the holo-form of the protein was present (i.e., carrying the 4'-phosphopantetheine prosthetic group), with the remainder (and 100% of the otc and fren samples) being in the apo-form. Increasing incubation time post heat induction led to an increase in act holo-ACP. The recombinant act and gra ACPs could function in vitro as substrates for an S. coelicolor malonyl CoA:ACP acyl transferase, as measured by the coupling of a labelled malonyl unit to the ACP; their quantitative abilities to do so correlated with the proportions of deduced holo form in the two samples.

Purification of a malonyltransferase from Streptomyces coelicolor A3(2) and analysis of its genetic determinant.

Streptomyces coelicolor A3(2) synthesizes each half molecule of the dimeric polyketide antibiotic actinorhodin (Act) from one acetyl and seven malonyl building units, catalyzed by the Act polyketide synthase (PKS). The synthesis is analogous to fatty acid biosynthesis, and there is evident structural similarity between PKSs of Streptomyces spp. and fatty acid synthases (FASs). Each system should depend on a malonyl coenzyme A:acyl carrier protein malonyltransferase, which charges the FAS or PKS with the malonyl units for carbon chain extension. We have purified the Act acyl carrier protein-dependent malonyltransferase from stationary-phase, Act-producing cultures and have determined the N-terminal amino acid sequence and cloned the structural gene. The deduced amino acid sequence resembles those of known malonyltransferases of FASs and PKSs. The gene lies some 2.8 Mb from the rest of the act cluster, adjacent to an open reading frame whose gene product resembles ketoacylsynthase III of Escherichia coli FAS. The malonyltransferase was expressed equally as well during vegetative growth (when other components of the act PKS were not expressed) as in the stationary phase, suggesting that the malonyltransferase may be shared between the FAS and PKS of S. coelicolor. Disruption of the operon containing the malonyltransferase gene proved to be impossible, supporting the idea that the malonyltransferase plays an essential role in fatty acid biosynthesis.

Cloning, sequencing, and analysis of the griseusin polyketide synthase gene cluster from Streptomyces griseus.

A fragment of DNA was cloned from the Streptomyces griseus K-63 genome by using genes (act) for the actinorhodin polyketide synthase (PKS) of Streptomyces coelicolor as a probe. Sequencing of a 5.4-kb segment of the cloned DNA revealed a set of five gris open reading frames (ORFs), corresponding to the act PKS genes, in the following order: ORF1 for a ketosynthase, ORF2 for a chain length-determining factor, ORF3 for an acyl carrier protein, ORF5 for a ketoreductase, and ORF4 for a cyclase-dehydrase. Replacement of the gris genes with a marker gene in the S. griseus genome by using a single-stranded suicide vector propagated in Escherichia coli resulted in loss of the ability to produce griseusins A and B, showing that the five gris genes do indeed encode the type II griseusin PKS. These genes, encoding a PKS that is programmed differently from those for other aromatic PKSs so far available, will provide further valuable material for analysis of the programming mechanism by the construction and analysis of strains carrying hybrid PKS.

Purification and separation of holo- and apo-forms of Saccharopolyspora erythraea acyl-carrier protein released from recombinant Escherichia coli by freezing and thawing.

Saccharopolyspora erythraea acyl-carrier protein, highly expressed from a T7-based expression plasmid in Escherichia coli, can be selectively released from the cells in near-quantitative yield by a single cycle of freezing and thawing in a neutral buffer. Electrospray mass spectrometry was used to confirm that the recombinant S. erythraea acyl-carrier protein over-expressed in E. coli is present predominantly as the holo-form, with variable amounts of apo-acyl-carrier protein, holo-acyl-carrier protein dimer and holo-acyl-carrier protein glutathione adduct. The holo- and apo-acyl-carrier proteins are both readily purified on a large scale from the freeze-thaw extracts and can be separated from one another by octyl-Sepharose chromatography. The holo-acyl-carrier protein obtained in this way was fully active in supporting the synthesis of acyl-acyl-carrier protein by extracts of S. erythraea.

Cloning, characterization, and high-level expression in Escherichia coli of the Saccharopolyspora erythraea gene encoding an acyl carrier protein potentially involved in fatty acid biosynthesis.

The erythromycin A-producing polyketide synthase from the gram-positive bacterium Saccharopolyspora erythraea (formerly Streptomyces erythraeus) has evident structural similarity to fatty acid synthases, particularly to the multifunctional fatty acid synthases found in eukaryotic cells. Fatty acid synthesis in S. erythraea has previously been proposed to involve a discrete acyl carrier protein (ACP), as in most prokaryotic fatty acid synthases. We have cloned and sequenced the structural gene for this ACP and find that it does encode a discrete small protein. The gene lies immediately adjacent to an open reading frame whose gene product shows sequence homology to known beta-ketoacyl-ACP synthases. A convenient expression system for the S. erythraea ACP was obtained by placing the gene in the expression vector pT7-7 in Escherichia coli. In this system the ACP was efficiently expressed at levels 10 to 20% of total cell protein. The recombinant ACP was active in promoting the synthesis of branched-chain acyl-ACP species by extracts of S. erythraea. Electrospray mass spectrometry is shown to be an excellent method for monitoring the efficiency of in vivo posttranslational modification of ACPs.