AGOS: A Plug-and-Play Method for the Assembly of Artificial Gene Operons into Functional Biosynthetic Gene Clusters.

The generation of novel secondary metabolites by reengineering or refactoring biochemical pathways is a rewarding but also challenging goal of synthetic biology. For this, the development of tools for the reconstruction of secondary metabolite gene clusters as well as the challenge of understanding the obstacles in this process is of great interest. The artificial gene operon assembly system (AGOS) is a plug-and-play method developed as a tool to consecutively assemble artificial gene operons into a destination vector and subsequently express them under the control of a de-repressed promoter in a Streptomyces host strain. AGOS was designed as a set of entry plasmids for the construction of artificial gene operons and a SuperCos1 based destination vector, into which the constructed operons can be assembled by Red/ET-mediated recombination. To provide a proof-of-concept of this method, we disassembled the well-known novobiocin biosynthetic gene cluster into four gene operons, encoding for the different moieties of novobiocin. We then genetically reorganized these gene operons with the help of AGOS to finally obtain the complete novobiocin gene cluster again. The production of novobiocin precursors and of novobiocin could successfully be detected by LC-MS and LC-MS/MS. Furthermore, we demonstrated that the omission of terminator sequences only had a minor impact on product formation in our system.

Mutational analysis of a phenazine biosynthetic gene cluster in Streptomyces anulatus 9663.

The biosynthetic gene cluster for endophenazines, i.e., prenylated phenazines from Streptomyces anulatus 9663, was heterologously expressed in several engineered host strains derived from Streptomyces coelicolor M145. The highest production levels were obtained in strain M512. Mutations in the rpoB and rpsL genes of the host, which result in increased production of other secondary metabolites, had no beneficial effect on the production of phenazines. The heterologous expression strains produced, besides the known phenazine compounds, a new prenylated phenazine, termed endophenazine E. The structure of endophenazine E was determined by high-resolution mass spectrometry and by one- and two-dimensional NMR spectroscopy. It represented a conjugate of endophenazine A (9-dimethylallylphenazine-1-carboxylic acid) and L-glutamine (L-Gln), with the carboxyl group of endophenazine A forming an amide bond to the α-amino group of L-Gln. Gene inactivation experiments in the gene cluster proved that ppzM codes for a phenazine N-methyltransferase. The gene ppzV apparently represents a new type of TetR-family regulator, specifically controlling the prenylation in endophenazine biosynthesis. The gene ppzY codes for a LysR-type regulator and most likely controls the biosynthesis of the phenazine core. A further putative transcriptional regulator is located in the vicinity of the cluster, but was found not to be required for phenazine or endophenazine formation. This is the first investigation of the regulatory genes of phenazine biosynthesis in Streptomyces.

Heterologous expression of the biosynthetic gene clusters of coumermycin A(1), clorobiocin and caprazamycins in genetically modified Streptomyces coelicolor strains.

The biosynthetic gene clusters of the aminocoumarin antibiotics clorobiocin and coumermycin A(1) and of the liponucleoside antibiotic caprazamycin were stably integrated into the genomes of different host strains derived from Streptomyces coelicolor A3(2). For the heterologous expression of clorobiocin derivatives in a chemically defined medium, inclusion of 0.6% of the siloxylated ethylene oxide/propylene oxide copolymer Q2-5247 into the growth medium proved to result in a 4.8-fold increase of productivity. Presumably, this copolymer acts as an oxygen carrier. The additional inclusion of cobalt chloride (0.2-2 mg l(-1)) dramatically increased the percentage of the desired compound clorobiocin within the total produced clorobiocin derivatives. This is very likely due to a stimulation of a cobalamin-dependent methylation reaction catalyzed by the enzyme CloN6 of clorobiocin biosynthesis. All three investigated host strains (S. coelicolor M512, M1146 and M1154) gave similar production rates of total clorobiocin derivatives (on average, 158 mg l(-1) in the presence of 0.6% Q2-5247 and 0.2 mg l(-1) CoCl(2)). In contrast, heterologous production of caprazamycin derivatives was optimal in strain M1154 (amounts of 152 mg l(-1) on average).

Use of an inducible promoter for antibiotic production in a heterologous host.

The biosynthetic gene cluster of the aminocoumarin antibiotic novobiocin comprises 20 coding sequences. Sixteen of them code for essential enzymes for novobiocin formation, transcribed in the form of a single 18-kb polycistronic mRNA. In the present study, we replaced the genuine promoter of this operon by the tetracycline-inducible promoter tcp830 and at the same time deleting the two pathway-specific positive regulator genes of novobiocin biosynthesis. The heterologous producer Streptomyces coelicolor M512 harboring the modified gene cluster produced, upon addition of 2 mg L(-1) of the inducer compound anhydrotetracyline, 3.4-fold more novobiocin than strains carrying the unmodified cluster. A second tcp830 promoter was inserted in the middle of the 18-kb operon in order to ensure adequate transcription of the rearmost genes. However, this did not lead to a further increase of novobiocin formation, showing that a single tcp830 promoter was sufficient to achieve high transcription of all 16 genes of the operon. A high induction of novobiocin formation was achieved within a wide range of anhydrotetracyline concentrations (0.25-2.0 mg L(-1)). Growth of the strains was not affected by these concentrations. The inducer compound could be added either at the time of inoculation or at any other time up to mid-growth phase, always achieving a similar final antibiotic production. Therefore, the tcp830 promoter presents a robust, easy-to-use system for the inducible expression of biosynthetic gene clusters in heterologous hosts, independent from the genuine regulatory network.

Use of a halogenase of hormaomycin biosynthesis for formation of new clorobiocin analogues with 5-chloropyrrole moieties.

The depsipeptide antibiotic hormaomycin, which is produced by Streptomyces griseoflavus W-384, contains a 5-chloropyrrole moiety. In the producer strain we identified the gene hrmQ that shows sequence similarity to FADH(2)-dependent halogenases. This gene was cloned and heterologously expressed in Streptomyces roseochromogenes var. oscitans DS12.976, which is the producer of the aminocoumarin antibiotic clorobiocin, which contains a 5-methylpyrrole moiety. For the present experiment, we used a mutant of this strain in which the respective pyrrole-5-methyltransferase had been inactivated. Expression of the halogenase hrmQ in this mutant strain led to the formation of two new clorobiocin derivatives that carried a 5-chloropyrrole moiety. These compounds were isolated on a preparative scale, their structures were elucidated by (1)H NMR spectroscopy and mass spectrometry, and their antibacterial activity was determined. The substrate of HrmQ is likely to be a pyrrole-2-carboxyl-S-[acyl carrier protein] thioester. If this assumption is true, this study presents the first experiment in combinatorial biosynthesis that uses a halogenase that acts on an acyl carrier protein-bound substrate.

A 7-dimethylallyltryptophan synthase from Aspergillus fumigatus: overproduction, purification and biochemical characterization.

A putative prenyltransferase gene, Afu3g12930, was identified in the genome sequence of Aspergillus fumigatus. EAL92290, encoded by Afu3g12930, consists of 472 aa, with a molecular mass of about 53 kDa. The coding sequence of Afu3g12930 was cloned in pQE60, and overexpressed in Escherichia coli. The soluble His(6)-fusion protein was purified to apparent homogeneity, and characterized biochemically. The enzyme was found to catalyse the prenylation of Trp at the C-7 position of the indole moiety, in the presence of dimethylallyl diphosphate (DMAPP); therefore, it functions as a 7-dimethylallyltryptophan synthase (7-DMATS). The structure of the enzymic product was elucidated by NMR and MS analysis. K(m) values were 67 microM for DMAPP, and 137 microM for l-Trp. Geranyl diphosphate was not accepted as prenyl donor, while Trp-containing dipeptides were found to be aromatic substrates of 7-DMATS. 7-DMATS did not need divalent metal ions for its enzymic reaction, although Ca(2+) enhanced the reaction velocity slightly. The enzyme is the second dimethylallyltryptophan synthase identified in A. fumigatus. Interestingly, it shares a sequence identity of only 31 % at the amino acid level with another known dimethylallyltryptophan synthase, FgaPT2, from the same fungus; FgaPT2 prenylates l-Trp at the C-4 position of the indole ring. Afu3g12930 belongs to a putative biosynthetic gene cluster consisting of eight genes. Orthologous clusters were also identified in the genome sequences of Neosartorya fischeri and Aspergillus terreus. The putative roles of the genes in the cluster are discussed.

A soluble, magnesium-independent prenyltransferase catalyzes reverse and regular C-prenylations and O-prenylations of aromatic substrates.

Fnq26 from Streptomyces cinnamonensis DSM 1042 is a new member of the recently identified CloQ/Orf2 class of prenyltransferases. The enzyme was overexpressed in E. coli and purified to apparent homogeneity, resulting in a soluble, monomeric protein of 33.2 kDa. The catalytic activity of Fnq26 is independent of the presence of Mg(2+) or other divalent metal ions. With flaviolin (2,5,7-trihydroxy-1,4-naphthoquinone) as substrate, Fnq26 catalyzes the formation of a carbon-carbon-bond between C-3 (rather than C-1) of geranyl diphosphate and C-3 of flaviolin, i.e. an unusual "reverse" prenylation. With 1,3-dihydroxynaphthalene and 4-hydroxybenzoate as substrates Fnq26 catalyzes O-prenylations.

CdpNPT, an N-prenyltransferase from Aspergillus fumigatus: overproduction, purification and biochemical characterisation.

A putative prenyltransferase gene, cdpNPT, was identified in the genome sequence of Aspergillus fumigatus by a homology search by using known prenyltransferases and sequence analysis. CdpNPT consists of 440 amino acids and has a molecular mass of about 50 kDa. The coding sequence of cdpNPT was cloned in pQE60 and overexpressed in E. coli. The soluble His(6)-fusion CdpNPT was purified to near homogeneity and characterised biochemically. The enzyme showed broad substrate specificity towards aromatic substrates and was found to catalyse the prenylation of tryptophan-containing cyclic dipeptides at N1 of the indole moieties in the presence of dimethylallyl diphosphate (DMAPP); geranyl diphosphate was not accepted as prenyl donor. The structures of the enzymatic products were elucidated by NMR and MS analysis. The K(m) value for DMAPP was determined to be 650 microM. Due to substrate inhibition, K(m) values could not be obtained for the aromatic substrates. CdpNPT does not need divalent metal ions for its enzymatic reaction, although Ca(2+) enhances the reaction velocity by up to the threefold. CdpNPT is the first N-prenyltransferase that has been purified and characterised in a homogenous form after heterologous overproduction. Interestingly, it shows significant sequence similarity to other indole prenyltransferases that catalyse the formation of C--C bonds.

CloN6, a novel methyltransferase catalysing the methylation of the pyrrole-2-carboxyl moiety of clorobiocin.

The aminocoumarin antibiotic clorobiocin contains a 5-methylpyrrole-2-carboxylic acid unit. This pyrrole unit is derived from L-proline, and it would be expected that its 5-methyl group should be introduced by a methylation reaction. However, sequence analysis of the clorobiocin biosynthetic gene cluster did not reveal a gene with sequence similarity to the SAM-dependent methyltransferases that could be assigned to this reaction. This study, however, has provided evidence that the gene cloN6 is involved in this methylation reaction. Its gene product CloN6 shares conserved sequence motifs with the recently identified radical SAM protein superfamily, and it has been suggested that members of this family can catalyse methylcobalamin-dependent methylation reactions. cloN6 was inactivated in the clorobiocin producer Streptomyces roseochromogenes var. oscitans DS 12.976 by use of the PCR-targeting method. The cloN6(-) mutants accumulated, instead of clorobiocin, a derivative lacking the 5"'-methyl group of the pyrrole moiety (termed novclobiocin 109). A structural isomer carrying the pyrrole-2-carboxyl moiety at 2"-OH rather than at the 3"-OH of the deoxysugar (novclobiocin 110), and a derivative completely lacking the pyrrole unit (novclobiocin 104) were also identified. The structures of the metabolites were confirmed by NMR and MS analysis. Antibacterial activity tests against Bacillus subtilis showed that novclobiocin 109 and novclobiocin 110 have antibacterial activities about eight times less than that of clorobiocin, whereas novclobiocin 104 showed no activity under the test conditions.

Methyltransferase genes in Streptomyces rishiriensis: new coumermycin derivatives from gene-inactivation experiments.

The coumarin antibiotic coumermycin A(1) contains at least eight methyl groups, presumably derived from S-adenosylmethionine. Two putative methyltransferase genes, couO and couP, of the coumermycin A(1) biosynthetic gene cluster were inactivated by in-frame deletion. In the resulting mutants, coumermycin A(1) production was abolished. New coumermycin derivatives were accumulated instead, and were identified by HPLC-MS using selected reaction monitoring via electrospray ionization. couO mutants accumulated a coumermycin derivative lacking the methyl groups at C-8 of the characteristic aminocoumarin rings, whereas in the couP mutant a coumermycin derivative lacking the methyl groups at the 4-hydroxyl groups of the two deoxysugar moieties was identified. These results provided evidence that couO encodes a C-methyltransferase responsible for the transfer of a methyl group to C-8 of the aminocoumarin ring, and couP an O-methyltransferase for methylation of 4-OH of the sugar in the biosynthesis of coumermycin A(1), respectively. C-methylation of the aminocoumarin ring is considered as an early step of coumermycin biosynthesis. Nevertheless, the intermediates with the non-methylated aminocoumarin ring were accepted by the enzymes catalysing the subsequent steps of the pathway. The new, demethylated secondary metabolites were produced in an amount at least as high as that of coumermycin A(1) in the wild-type.