Engineered biosynthesis of novel polyketides.

Polyketide synthases (PKSs) are multifunctional enzymes that catalyze the biosynthesis of a huge variety of carbon chains differing in their length and patterns of functionality and cyclization. Many polyketides are valuable therapeutic agents. A Streptomyces host-vector system has been developed for efficient construction and expression of recombinant PKSs. Using this expression system, several novel compounds have been synthesized in vivo in significant quantities. Characterization of these metabolites has provided new insights into key features of actinomycete aromatic PKS specificity. Thus, carbon chain length is dictated, at least in part, by a protein that appears to be distinctive to this family of PKSs, whereas the acyl carrier proteins of different PKSs can be interchanged without affecting product structure. A given ketoreductase can recognize and reduce polyketide chains of different length; this ketoreduction always occurs at the C-9 position. The regiospecificity of the first cyclization of the nascent polyketide chain is either determined by the ketoreductase, or the chain-extending enzymes themselves. However, the regiospecificity of the second cyclization is determined by a distinct cyclase, which can discriminate between substrates of different chain lengths.

Engineered biosynthesis of novel polyketides: properties of the whiE aromatase/cyclase.

The ORFVI from the cluster of genes, which is responsible for the biosynthesis of the Streptomyces coelicolor spore pigment, the whiE cluster, has been described as a bifunctional aromatase/cyclase. In order to evaluate its potential use for generating novel polyketides, combinations of this gene with those encoding minimal polyketide synthase enzymes with or without a ketoreductase from S. coelicolor A3(2) were constructed and analyzed in vivo. Analysis of the polyketide products generated from these constructs indicates that the whiE-ORFVI enzyme has properties similar to those of TcmN, although the whiE aromatase/cyclase normally acts on a polyketide intermediate that is four carbons longer than the TcmN substrate. The whiE aromatase/cyclase can influence the regiospecificity of the first cyclization of unreduced, but not reduced, backbones and is also responsible for the second ring aromatization. An unusual new polyketide, EM18, was identified which is not seen in equivalent strains expressing the tcmN aromatase/cyclase or the act aromatase genes. The structure of EM18 suggests that the WhiE-ORFVI product might have some unique properties within this family of polyketide synthase subunits, and may therefore be useful in the design of combinatorial biosynthetic strategies.

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.

Engineered biosynthesis of novel polyketides: evidence for temporal, but not regiospecific, control of cyclization of an aromatic polyketide precursor.

BACKGROUND: Aromatic polyketide synthases (PKSs) catalyze the formation and cyclization of polyketide chains of variable lengths, generating a family of compounds of proven medical significance. Initial control over the regiospecificity of cyclization is believed to be exercised by the minimal PKS, composed of the three essential components for polyketide biosynthesis, which catalyzes an intramolecular aldol condensation towards the middle of the chain. Subsequent cyclization reactions are either catalyzed by additional components of the PKS, or occur in the absence of specific catalysts. RESULTS: Structural and biosynthetic studies on SEK4b, a novel octaketide product of a minimal PKS, revealed an unusual cyclization pattern. The first cyclization (an aldol condensation) occurs at the methyl end of the unreduced polyketide backbone precursor. This is followed by hemiketal formation and lactonization. The overall structure of SEK4b is similar to that of SEK4, a previously-identified product of the same genetically-engineered strain, differing only in the positions of a methyl and a pyrone group around a common fused-ring system. The biosynthetic pathways of the two molecules are quite different, however. The yield of SEK4b relative to SEK4 is much higher in the absence of PKS components (aromatases and cyclases) acting later in the pathway. CONCLUSIONS: In this cyclization pathway, the regiospecificity of cyclization is not directly controlled by the minimal PKS. Instead, we propose that the enzyme influences cyclization by controlling the timing of chain release. Chain release and cyclization may be concurrent with synthesis. Other PKS subunits appear to stabilize the complex of the PKS with the nascent chain, preventing premature release.

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.

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.

Genetic engineering of Streptomyces to create hybrid antibiotics.

Over the past year, dramatic developments in the technology for isolating and manipulating genes for polyketide synthases have been reported. Significant progress has been made in understanding the mechanisms by which these complex enzymes generate the carbon chains of the polyketides, a highly versatile class of natural products. With the demonstration of the production of novel metabolites by synthase engineering, the stage is excitingly set for rationally manipulating synthase 'programming' to generate tailor-made carbon chains.

Cloning and expression of a p-aminobenzoic acid synthetase gene of the candicidin-producing Streptomyces griseus.

4.5-kb BamHI fragments of DNA coding for p-aminobenzoic acid (PABA) synthetase from the candicidin-producing Streptomyces griseus IMRU 3570 and from a sulphonamide resistant mutant of it were cloned on the plasmid vector pIJ41 into Streptomyces lividans 66. The cloned DNA restored prototrophy to a pab auxotroph of S. lividans; when the S. griseus donor was a sulphonamide resistant, PABA-overproducing mutant, the S. lividans clone was sulphonamide resistant as well as Pab+. Sub-cloning the 4.5-kb fragment of S. griseus DNA into Escherichia coli pabA- or pabB- mutants by insertion at the BamHI site of pBR322 did not yield prototrophic clones directly. However, when the cloned fragment had the proper orientation relative to the tet promoter, but not the opposite one, it was possible to select Pab+ colonies, which arose by deletion in vivo of approx. 1 kb of the S. griseus inserted DNA. These results, and those of studies in which Tn5 abolished the Pab+ phenotype by insertion in vivo in the tet promoter or downstream of it, indicated that the S. griseus pab promoter was not expressed in E. coli but that the pab gene could be expressed by transcriptional readthrough from the vector. Experiments in which the cloned DNA was transferred back from E. coli to S. lividans suggested, but did not prove, that the Streptomyces pab promoter had been deleted by loss of the approx. 1-kb segment. These experiments showed expression of both the tet (of pBR322) and kan (of Tn5) promoters in S. lividans.

Nucleotide sequence, transcription and deduced function of a gene involved in polyketide antibiotic synthesis in Streptomyces coelicolor.

The BamHI fragment containing the actIII gene, from the actinorhodin (Act) biosynthetic gene cluster of Streptomyces coelicolor A3(2), was sequenced. The derived amino acid sequence for the actIII gene shows homology to known oxidoreductases and the actIII product is believed to be responsible for catalysing a beta-keto reductive step during assembly of the Act polyketide chain. High resolution transcript mapping identified the transcription start point at 33 nucleotides upstream of the putative translation start codon. The transcript ends in a large invertedly repeated sequence. In vivo promoter-probe studies suggest that efficient transcription of the actIII gene requires the product of the actII gene.

Expression of a Streptomyces plasmid promoter in Escherichia coli.

A 166-bp DNA fragment from the Streptomyces multicopy plasmid pIJ101 with in vivo promoter activity both in Streptomyces lividans and in Escherichia coli was isolated. The start point of the RNA transcribed from this fragment, determined by high resolution S1 nuclease mapping, was the same in S. lividans and in E. coli. This suggests that the E. coli RNA polymerase recognizes the same sequence determinants and chooses the point of initiation of RNA synthesis in the same way as the corresponding S. lividans enzyme. The putative promoter sequence shows good homology to the E. coli promoter consensus sequence in the '-35' region but poor homology in the '-10' region.

The Streptomyces plasmid SCP2*: its functional analysis and development into useful cloning vectors.

Detailed restriction maps of the plasmid SCP2* and its deletion derivative pSCP103 were constructed. DNA fragments carrying hygromycin (Hyg), thiostrepton (Thio) or viomycin-resistance (VioR) determinants were inserted into pSCP103, and various segments were deleted from the resulting plasmids. Changes in plasmid phenotypes associated with these insertions and deletions allowed the localisation and characterisation of plasmid replication, stability, transfer and fertility functions. Several useful cloning vectors were constructed. They are able to maintain large (greater than 30 kb) DNA inserts, with stable inheritance at a low copy number (1-2 per chromosome) and without structural rearrangements, in Streptomyces hosts. The vectors have a broad host range in the genus Streptomyces. One of them (pIJ903) is a shuttle vector for Streptomyces and Escherichia coli.

Cloning of a chloramphenicol acetyltransferase gene of Streptomyces acrimycini and its expression in Streptomyces and Escherichia coli.

A gene (cat) for chloramphenicol (Cm) acetyltransferase (CAT) was cloned from Streptomyces acrimycini into S. lividans 66 on the plasmid vector pIJ61. The cat gene was localized on a 1.7-kb BclI fragment, which probably also carries the cat promoter. This DNA fragment conferred Cm resistance, through CAT activity, on S. lividans, S. coelicolor and S. parvulus, but not on Escherichia coli when inserted in the BamHI site of the tetracycline-resistance(TcR) gene of pBR322. However, when inserted in a particular orientation in this site, spontaneous deletions of 0.7 kb led to CAT activity and Cm resistance. DNA homologous to the 1.7-kb BclI cat fragment was found in most, but not all, of a series of other streptomycetes that have CAT activity. The cat provides a potentially useful screening marker for Streptomyces cloning vectors.

Cloning and expression of a puromycin N-acetyl transferase gene from Streptomyces alboniger in Streptomyces lividans and Escherichia coli.

A gene (pac) encoding a puromycin N-acetyl transferase (PAC) of Streptomyces alboniger ATCC12461 was cloned in the Streptomyces plasmid pIJ702 and expressed in S. lividans 1326. Several clones resistant to puromycin were isolated and shown to carry pIJ702 with different inserts of S. alboniger DNA. They were classified as of low and high activity according to the levels of enzymatic activity expressed by them. The different levels of expression were related to the two possible orientations of the S. alboniger DNA inserts in the pIJ702 vector. Six of the recombinant plasmids contain a common 1.6-kb DNA sequence which, by subcloning experiments, was shown to carry a pac gene encoding PAC activity. The pac gene was subcloned next to the lac promoter of Escherichia coli plasmid pUC19. Only one of the two possible orientations of insertion expressed PAC activity, suggesting that it was dependent on the lac promoter. Accordingly, isopropylthio-beta-D-galactoside (IPTG) was able to stimulate the expression of the enzyme activity. These results allowed the direction of transcription of the pac gene to be determined.

Physical analysis of antibiotic-resistance genes from Streptomyces and their use in vector construction.

Restriction endonuclease cleavage maps of five DNA fragments carrying genes for neomycin phosphotransferase and neomycin acetyltransferase (from Streptomyces fradiae), viomycin phosphotransferase (from S. vinaceus), and ribosomal methylases determining resistance to thiostrepton (from S. azureus) and MLS antibiotics (from S. erythreus) are described, together with a map for the SLP1.2 Streptomyces plasmid used to isolate the fragments. Construction of a versatile Streptomyces cloning vector (pIJ61) is reported. pIJ61 carries neomycin phosphotransferase and thiostrepton resistance genes and has unique BamHI and PstI sites which will allow clone recognition by insertional inactivation of neomycin resistance; cloning sites for several other endonucleases are also present. pIJ28, a shuttle vector for Streptomyces and E. coli, carries neomycin resistance and the SLP1.2 and pBR322 replicons.

Cloning and expression in a heterologous host of the complete set of genes for biosynthesis of the Streptomyces coelicolor antibiotic undecylprodigiosin.

A fragment of DNA carrying the hitherto unisolated members of the cluster of genes (red) for biosynthesis of the red-pigmented antibiotic undecylprodigiosin of Streptomyces coelicolor A3(2) was isolated. This was done by cloning random fragments of S. coelicolor DNA into the closely related Streptomyces lividans 66 and recovering a clone that caused overproduction of undecylprodigiosin. The effect was probably due to the presence of the cloned redD gene, which functions as a positive regulator of the expression of the red cluster, activating the normally poorly expressed red genes of S. lividans. Two fragments from either end of the red cluster were cloned adjacent to each other on a low-copy-number Streptomyces vector. Double crossing-over occurring between these plasmid-borne sequences and the chromosomal copy of the same DNA in S. coelicolor led to isolation of the entire red cluster as a single cloned fragment. Isolation of antibiotic biosynthetic genes by the effects of an activator in a self-cloning experiment, and in vivo reconstitution of a large cluster of genes by homologous recombination, may turn out to be usefully generalizable procedures.

Cloning, sequencing and deduced functions of a cluster of Streptomyces genes probably encoding biosynthesis of the polyketide antibiotic frenolicin.

A 10.2-kb fragment of DNA from Streptomyces roseofulvus, which contains polyketide synthase (PKS)-encoding genes (fren) presumed to determine production of the antibiotics frenolicin and the nanaomycins, was cloned. A 5530-bp continuous segment of this DNA was sequenced. Analysis of the sequence revealed five complete open reading frames (ORFs) transcribed in one direction (ORFs 1, 2, 3, 5, 4) and one (ORFX), located between ORF3 and ORF5, transcribed in the opposite direction. The deduced amino-acid sequences of ORFs 1, 2, 3, 4 and 5 closely resemble the sequences of known components of the type-II PKS from other Streptomyces species: putative heterodimeric (ORF1 + 2) ketosynthase, acyl carrier protein, cyclase and ketoreductase, respectively. A resemblance between the N-terminal and C-terminal halves of the ORF4 product--also discovered in the corresponding genes from other isochromanequinone antibiotic producers--suggests a possible origin of the cyclase-encoding gene by duplication. ORFX appears to represent a novel class of genes of unknown function present not only in the fren cluster, but also in other clusters of aromatic antibiotic biosynthetic genes in Streptomyces species. The fren-ORF1-5 genes, encoding a PKS that constructs a nascent polyketide of either 16 or 18 carbons, compared with fixed lengths of 16 and 20 for other available examples, are proving to be valuable for understanding the mechanisms controlling polyketide chain length and patterns of reduction and cyclisation.

Characterization of two groEL genes in Streptomyces coelicolor A3(2).

Two Streptomyces coelicolor A3(2) groEL-like genes, groEL1 and groEL2, were cloned and characterized. Pulsed-field-gel electrophoresis located these genes, which were not adjacent, in the same segment of the chromosome. Nucleotide sequence analysis revealed that groEL1, but not groEL2, was preceded by a groES-like gene. Northern blots showed that heat shock induced the accumulation of transcripts corresponding to groES (0.7 kb), groES/EL1 (2.3 kb) and groEL2 (2.1 kb). Unique transcription start points and promoters were located for groES/EL1 and groEL2, having -10 and -35 hexamers similar to eubacterial vegetative promoters. Regions located 5' to the groES/EL1 or groEL2 structural genes contain 'GCACTCN9GAGTGC' motifs conserved upstream from the heat-shock genes of other bacteria.

Nucleotide sequence of the chloramphenicol acetyltransferase gene of Streptomyces acrimycini.

The nucleotide sequence of a gene (cat) encoding chloramphenicol acetyltransferase (CAT) in Streptomyces acrimycini was determined. The predicted amino acid sequence demonstrates extensive homology with those of CATs isolated from Gram-negative Enterobacteria, notably with the type III variant encoded by the IncK plasmid R387. Transcript mapping indicates a single cat mRNA with a 5' end coinciding with the AUG codon used for translational initiation in vivo. We also determined the extent of a spontaneous deletion in the 5'-noncoding DNA, which occurs when the gene is cloned in the BamHI site of pBR322 in a specific orientation and which results in constitutive cat expression in Escherichia coli from the tet promoter.

Sequences of the oxytetracycline polyketide synthase-encoding otc genes from Streptomyces rimosus.

The complete nucleotide sequences of the Streptomyces rimosus oxytetracycline (oxyTc) polyketide synthase (PKS)-encoding genes (otcY) has been determined, revealing three open reading frames. The deduced amino-acid sequences correspond to the presumed heterodimeric beta-ketoacyl synthase and acyl carrier protein found in other type-II (multicomponent) PKS systems that specify construction of acetate-derived polyketide antibiotics.

Conserved secondary structure in the actinorhodin polyketide synthase acyl carrier protein from Streptomyces coelicolor A3(2) and the fatty acid synthase acyl carrier protein from Escherichia coli.

The acyl carrier protein (ACP) of Streptomyces coelicolor A3(2) functions as a molecular chaperone during the biosynthesis of the polyketide actinorhodin (act). Here we compare structural features of the polyketide synthase (PKS) ACP, determined by two-dimensional 1H-NMR, with the Escherichia coli fatty acid synthase (FAS) ACP. The PKS ACP contains four helices (residues 7-16 [A], 42-53 [B], 62-67 [C], 72-86 [D]), and a large loop (residues 17-41) having no defined secondary structure with the exception of a turn between residues 21 and 24. The act ACP shows 47% sequence similarity with the E. coli FAS ACP and the results demonstrate that the sequence homology is extended to the secondary structure of the proteins.