Engineering a polyketide with a longer chain by insertion of an extra module into the erythromycin-producing polyketide synthase.

BACKGROUND: Modular polyketide synthases catalyse the biosynthesis of medically useful natural products by stepwise chain assembly, with each module of enzyme activities catalysing a separate cycle of polyketide chain extension. Domain swapping between polyketide synthases leads to hybrid multienzymes that yield novel polyketides in a more or less predictable way. No experiments have so far been reported which attempt to enlarge a polyketide synthase by interpolating additional modules. RESULTS: We describe here the construction of tetraketide synthases in which an entire extension module from the rapamycin-producing polyketide synthase is covalently spliced between the first two extension modules of the erythromycin-producing polyketide synthase (DEBS). The extended polyketide synthases thus formed are found to catalyse the synthesis of specific tetraketide products containing an appropriate extra ketide unit. Co-expression in Saccharopolyspora erythraea of the extended DEBS multienzyme with multienzymes DEBS 2 and DEBS 3 leads to the formation, as expected, of novel octaketide macrolactones. In each case the predicted products are accompanied by significant amounts of unextended products, corresponding to those of the unaltered DEBS PKS. We refer to this newly observed phenomenon as 'skipping'. CONCLUSIONS: The strategy exemplified here shows far-reaching possibilities for combinatorial engineering of polyketide natural products, as well as revealing the ability of modular polyketide synthases to 'skip' extension modules. The results also provide additional insight into the three-dimensional arrangement of modules within these giant synthases.

Molecular basis of Celmer's rules: role of the ketosynthase domain in epimerisation and demonstration that ketoreductase domains can have altered product specificity with unnatural substrates.

BACKGROUND: Polyketides are structurally diverse natural products with a range of medically useful activities. Non-aromatic bacterial polyketides are synthesised on modular polyketide synthase multienzymes (PKSs) in which each cycle of chain extension requires a different 'module' of enzymatic activities. Attempts to design and construct modular PKSs that synthesise specified novel polyketides provide a particularly stringent test of our understanding of PKS structure and function. RESULTS: We show that the ketoreductase (KR) domains of modules 5 and 6 of the erythromycin PKS, housed in the multienzyme subunit DEBS3, exert an unexpectedly low level of stereochemical control in reducing the keto group of a synthetic analogue of the diketide intermediate. This led us to construct a hybrid triketide synthase based on DEBS3 with ketosynthase domain ketosynthase (KS)5 replaced by the loading module and KS1. The construct in vivo produced two major triketide stereoisomers, one expected and one surprising. The latter was of opposite configuration at three out of the four chiral centres: the branching alkyl centre was that produced by KS1 and, surprisingly, both hydroxyl centres produced by the reduction steps carried out by KR5 and KR6 respectively. CONCLUSIONS: These results demonstrate that the epimerising activity associated with module 1 of the erythromycin PKS can be conferred on module 5 merely by transfer of the KS1 domain. Moreover, the normally precise stereochemical control observed in modular PKSs is lost when KR5 and KR6 are challenged by an unfamiliar substrate, which is much smaller than their natural substrates. This observation demonstrates that the stereochemistry of ketoreduction is not necessarily invariant for a given KR domain and underlines the need for mechanistic understanding in designing genetically engineered PKSs to produce novel products.

Design and utility of oligonucleotide gene probes for fungal polyketide synthases.

BACKGROUND: Recent advances in the molecular biology of polyketide biosynthesis have allowed the engineering of polyketide synthases and the biological ('combinatorial') synthesis of novel polyketides. Additional structural diversity in these compounds could be expected if more diverse polyketide synthases (PKS) could be utilised. Fungal polyketides are highly variable in structure, reflecting a potentially wide range of differences in the structure and function of fungal PKS complexes. Relatively few fungal synthases have been investigated, perhaps because of a lack of suitable genetic techniques available for the isolation and manipulation of gene clusters from diverse hosts. We set out to devise a general method for the detection of specific PKS genes from fungi. RESULTS: We examined sequence data from known fungal and bacterial polyketide synthases as well as sequence data from bacterial, fungal and vertebrate fatty acid synthases in order to determine regions of high sequence conservation. Using individual domains such as beta-ketoacylsynthases (KS), beta-ketoreductases (KR) and methyltransferases (MeT) we determined specific short (ca 7 amino acid) sequences showing high conservation for particular functional domains (e.g. fungal KR domains involved in producing partially reduced metabolites; fungal KS domains involved in the production of highly reduced metabolites etc.). Degenerate PCR primers were designed matching these regions of specific homology and the primers were used in PCR reactions with fungal genomic DNA from a number of known polyketide producing species. Products obtained from these reactions were sequenced and shown to be fragments from as-yet undiscovered PKS gene clusters. The fragments could be used in blotting experiments with either homologous or heterologous fungal genomic DNA. CONCLUSIONS: A number of sequences are presented which have high utility for the discovery of novel fungal PKS gene clusters. The sequences appear to be specific for particular types of fungal polyketide (i.e. non-reduced, partially reduced or highly reduced KS domains). We have also developed primers suitable for amplifying segments of fungal genes encoding polyketide C-methyltransferase domains. Genomic fragments amplified using these specific primer sequences can be used in blotting experiments and have high potential as aids for the eventual cloning of new fungal PKS gene clusters.

Novel octaketide macrolides related to 6-deoxyerythronolide B provide evidence for iterative operation of the erythromycin polyketide synthase.

BACKGROUND: The macrolide antibiotic erythromycin A, like other complex aliphatic polyketides, is synthesised by a bacterial modular polyketide synthase (PKS). Such PKSs, in contrast to other fatty acid and polyketide synthases which work iteratively, contain a separate set or module of enzyme activities for each successive cycle of polyketide chain extension, and the number and type of modules together determine the structure of the polyketide product. Thus, the six extension modules of the erythromycin PKS (DEBS) together catalyse the production of the specific heptaketide 6-deoxyerythronolide B. RESULTS: A mutant strain of the erythromycin producer Saccharopolyspora erythraea, which accumulates the aglycone intermediate erythronolide B, was found unexpectedly to produce two novel octaketides, both 16-membered macrolides. These compounds were detectable in fermentation broths of wild-type S. erythraea, but not in a strain from which the DEBS genes had been specifically deleted. From their structures, both of these octaketides appear to be aberrant products of DEBS in which module 4 has 'stuttered', that is, has catalysed two successive cycles of chain extension. CONCLUSIONS: The isolation of novel DEBS-derived octaketides provides the first evidence that an extension module in a modular PKS has the potential to catalyse iterative rounds of chain elongation like other type I FAS and PKS systems. The factors governing the extent of such 'stuttering' remain to be determined.

Characterization of metabolites in intact Streptomyces citricolor culture supernatants using high-resolution nuclear magnetic resonance and directly coupled high-pressure liquid chromatography-nuclear magnetic resonance spectroscopy.

A novel NMR spectroscopic approach to the direct biochemical characterization of bacterial culture broths is presented. A variety of one- and two-dimensional 1H NMR spectroscopic methods were used to characterize low-molecular-weight organic components of broth supernatants from cultures of Streptomyces citricolor. By applying 1H NMR spectroscopy to analyze whole, untreated culture supernatants, it was possible to identify and monitor simultaneously a range of media substrates and excreted metabolites. Identified metabolites include 2-phenylethylamine, trehalose, succinate, acetate, uridine, and aristeromycin, a secondary metabolite with antibiotic properties. Directly coupled HPLC-NMR spectroscopy was also applied to the analysis of broth supernatants for the first time, to aid spectral assignments, especially where signals were extensively overlapped in the 1H NMR spectra of the whole broth mixtures. Two-dimensional NMR methods such as 1H-1H correlation spectroscopy, 1H-13C heteronuclear single quantum correlation, and 1H-13C heteronuclear multiple bond correlation aided the structure elucidation and peak assignments of individual components in the mixtures by providing information on 1H-1H coupling networks and 13C chemical shifts. This work shows that high-resolution NMR spectroscopic methods provide a rapid and efficient means of investigating microbial metabolism directly without invasive or destructive sample pretreatment.

Molecular basis of Celmer's rules: the role of two ketoreductase domains in the control of chirality by the erythromycin modular polyketide synthase.

BACKGROUND: Polyketides are compounds that possess medically significant activities. The modular nature of the polyketide synthase (PKS) multienzymes has generated interest in bioengineering new PKSs. Rational design of novel PKSs, however, requires a greater understanding of the stereocontrol mechanisms that operate in natural PKS modules. RESULTS: The N-acetyl cysteamine (NAC) thioester derivative of the natural beta-keto diketide intermediate was incubated with DEBS1-TE, a derivative of the erythromycin PKS that contains only modules 1 and 2. The reduction products of the two ketoreductase (KR) domains of DEBS1-TE were a mixture of the (2S, 3R) and (2R,3S) isomers of the corresponding beta-hydroxy diketide NAC thioesters. Repeating the incubation using a DEBS1-TE mutant that only contains KR1 produced only the (2S,3R) isomer. CONCLUSIONS: In contrast with earlier results, KR1 selects only the (2S) isomer and reduces it stereospecifically to the (2S, 3R)-3-hydroxy-2-methyl acyl product. The KR domain of module 1 controls the stereochemical outcome at both methyl-and hydroxyl-bearing chiral centres in the hydroxy diketide intermediate. Earlier work showed that the normal enzyme-bound ketoester generated in module 2 is not epimerised, however. The stereochemistry at C-2 is therefore established by a condensation reaction that exclusively gives the (2R)-ketoester, and the stereo-chemistry at C-3 by reduction of the keto group. Two different mechanisms of stereochemical control, therefore, operate in modules 1 and 2 of the erythromycin PKS. These results should provide a more rational basis for designing hybrid PKSs to generate altered stereochemistry in polyketide products.

Isolation and characterisation of an antifungal antibiotic (GR135402) with protein synthesis inhibition.

A novel antifungal antibiotic GR135402 has been isolated from a fermentation broth of Graphium putredinis which inhibited protein synthesis in Candida albicans but not rabbit reticulocytes. The spectrum of activity included C. albicans and Cryptococcus neoformans but not some other Candida species or Aspergillus species. Therapeutic efficacy in a mouse model of systemic candidosis was attained following parenteral dosing.

Induction of phenazine biosynthesis in cultures of Pseudomonas aeruginosa by L-N-(3-oxohexanoyl)homoserine lactone.

A range of Pseudomonas spp. and other Gram-negative bacteria were screened for induction of antimicrobial activity in response to the autoregulatory factor L-N-(3-oxohexanoyl)homoserine lactone. In one of these, P. aeruginosa ATCC 10145, the production of phenazine metabolites was shown to be inducible in a dose-dependent manner. The production of phenazine-1-carboxamide increased over 50-fold compared to control cultures when supplemented with 200 micrograms/ml of the autoregulator. In addition, the production of an unidentified polar antibacterial substance by this strain increased with autoregulator concentration.

Cloning and expression in Escherichia coli of a Streptomyces coelicolor A3(2) argCJB gene cluster.

From a partial Sau3AI library of Streptomyces coelicolor A3(2) DNA in pIJ916, two hybrid plasmids pGX1 and pGX2 were isolated that complemented S. coelicolor A3(2) or S. lividans arginine auxotrophs. Subcloning DNA from pGX1 in the Escherichia coli expression vector pRK9 containing the Serratia marcescens trp promoter gave rise to one plasmid, pZC2, that complemented E. coli argB, C, E and H auxotrophs, and another, pZC1, that complemented only the first three. The plasmids were markedly unstable in the various complemented hosts, to varying extents; pZC1 was characterized further as providing the stablest host/plasmid combinations. In vitro deletion of part of the vector's trp promoter did not affect complementation of the argB and C auxotrophs, implying that the S. coelicolor A3(2) arg genes may be expressed from their own promoter. The trp promoter-less plasmids included isolates, such as pZC177, that had suffered extensive further deletion without loss of complementing ability. Extracts of an E. coli argE auxotroph carrying pZC177 showed ornithine acetyltransferase activity, indicating that the complementing gene is of the argJ type. The complementation properties of in vitro deletion derivatives of pZC177 indicated the gene order argC-J-B. Part of argC and the upstream region were sequenced; an ORF was identified whose predicted product showed appreciable homology with the E. coli and Bacillus subtilis ArgC polypeptide. Upstream of this ORF a consensus-type promoter and ribosome binding site could be discerned; overlapping its promoter was a sequence with homology to arginine operators in these two other organisms.(ABSTRACT TRUNCATED AT 250 WORDS)

The squalestatins, novel inhibitors of squalene synthase produced by a species of Phoma. III. Biosynthesis.

The biosynthetic origin of the carbon and oxygen atoms of the novel fungal secondary metabolite 1 was studied. Incorporation studies with single and multiple labelled 13C precursors indicated that the major portion of the molecule was derived from two polyketide chains made up of acetate units. One of the chains had benzoic acid (which can be derived from phenylalanine) as a starter unit. The remaining carbons were derived from a four-carbon unit related to succinate and from methionine. Studies with [1-(13)C,18O2]acetate and 18O2 indicated that five of the oxygens, including both of the heterocyclic oxygens, were derived from atmospheric oxygen. The oxygens at the two ester carbonyls were derived from acetate.

Plasmid effects on secondary metabolite production by a streptomycete synthesizing an anthelmintic macrolide.

Transformation of the thermotolerant streptomycete, soil isolate S541, with plasmid cloning vectors of varying size, copy number, and parent replicon (derived from pIJ101, SCP2* and SLP1.2) depressed the biosynthesis of nemadectins (polyketide-derived secondary metabolites possessing anthelmintic activity). However, production of the chemically distinct 21-hydroxyl-oligomycin A, also produced by S541, was either unaffected or increased in plasmid-containing strains. A causal relationship between plasmid carriage and the changes in secondary metabolite yield was confirmed since cured strains were restored to normal production levels and their subsequent retransformation by plasmid DNA was followed by the same effects on nemadectin and oligomycin biosynthesis as before. All the plasmids tested were highly unstable in S541 and it was generally necessary to include an appropriate selective antibiotic (usually thiostrepton) in the growth medium. Thiostrepton was not responsible for the depressive effect, since this was also observed in plasmid-containing strains (i) when grown in antibiotic-free media and (ii) when alternative selective antibiotics such as neomycin were used. In addition, the plasmid-free strain produced both nemadectins and 21-hydroxyl-oligomycin A in the presence of sub-inhibitory levels of thiostrepton. The thiostrepton resistance gene, which was present on many of the plasmids tested, did not mediate the effect since plasmids carrying other selectable markers (pIJ58, neomycin, and pIJ355, viomycin) also depressed nemadectin but not 21-hydroxyl-oligomycin A production. No obvious recombination or integration events between S541 chromosomal DNA and any of the plasmids tested were revealed by DNA-DNA Southern hybridization.

Biosynthesis of the antibiotic actinorhodin. Analysis of blocked mutants of Streptomyces coelicolor.

From two types of class V act mutants of Streptomyces coelicolor two monomeric precursors of actinorhodin have been isolated and their structures determined. One is the known antibiotic kalafungin and the other a new compound. Their relationship to actinorhodin biosynthesis is discussed.

Production of 'hybrid' antibiotics by genetic engineering.

The recent development of molecular cloning systems in Streptomyces has made possible the isolation of biosynthetic genes for some of the many antibiotics produced by members of this important genus of bacteria. Such clones can now be used to test the idea that novel antibiotics could arise through the transfer of biosynthetic genes between streptomycetes producing different antibiotics. The likelihood of a 'hybrid' compound being produced must depend on the substrate specificities of the biosynthetic enzymes, about which little is known. In attempts to demonstrate hybrid antibiotic production, we therefore began with strains producing different members of the same chemical class of compounds in order to maximize the chance of success. Here we report the production of novel compounds by gene transfer between strains producing the isochromanequinone antibiotics actinorhodin, granaticin and medermycin. These experiments were made possible by the recent cloning of the whole set of genes for the biosynthetic pathway of actinorhodin from Streptomyces coelicolor A3(2) (ref. 8). We believe that this represents the first report of the production of hybrid antibiotics by genetic engineering.

A new channel-forming antibiotic from Streptomyces coelicolor A3(2) which requires calcium for its activity.

A recently discovered antibiotic (CDA; calcium-dependent antibiotic) of Streptomyces coelicolor A3(2) was found to be effective against a wide range of Gram-positive bacteria only in the presence of calcium ions. Producer and non-producer strains were identified and several media tested for their ability to support antibiotic production. The action of calcium was not simulated by any of the other cations tested. The antibiotic was found to induce discrete conductance fluctuations in planar lipid bilayer consistent with a channel-forming action. The electrical potential difference caused by a concentration difference of various salts across the CDA-containing bilayer, showed the channel to be cation-selective but of a size that discriminated against tetramethyl ammonium and choline ions. The data indicate that the antibiotic activity of CDA is due to its action as a calcium-dependent ionophore.

A pigmented mycelial antibiotic in Streptomyces coelicolor: control by a chromosomal gene cluster.

Streptomyces coelicolor was found to produce a third secondary metabolite, in addition to the antibiotics methylenomycin A and actinorhodin previously described. This is a red pigmented, highly non-polar compound with antibiotic activity against certain Gram-positive bacteria. Mutants lacking the red compound fell into five cosynthetic classes. Representatives of each of the five classes were mapped to the chromosome of the producing organism, in a closely linked cluster. Genetic studies provided evidence that this new metabolite is distinct from actinorhodin and indicated that the two pigments do not share parts of the same biosynthetic pathway.

Genetics of actinorhodin biosynthesis by Streptomyces coelicolor A3(2).

A series of 76 mutants of Streptomyces coelicolor A3(2) specifically blocked in the synthesis of the binaphthoquinone antibiotic actinorhodin were classified into seven phenotypic classes on the basis of antibiotic activity, accumulation of pigmented precursors or shunt products of actinorhodin biosynthesis, and cosynthesis of actinorhodin in pairwise combinations of mutants. The polarity of cosynthetic reactions, and other phenotypic properties, allowed six of the mutant classes to be arranged in the most probable linear sequence of biosynthetic blocks. One member of each mutant class was mapped unambigiguously to the chromosomal linkage map in the short segment between the hisD and guaA loci, suggesting that structural genes for actinorhodin biosynthesis may form an uninterrupted cluster of chromosomal genes.