DOI: 10.1002/cbic.201500048
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Functional Characterization of Different ORFs Including Luciferase-Like Monooxygenase Genes from the Mensacarcin Gene Cluster Sarah Maier,[a] Tanja Heitzler,[a] Katharina Asmus,[a] Elke Brçtz,[b, c] Uwe Hardter,[a] Katharina Hesselbach,[a] Thomas Paululat,[b] and Andreas Bechthold*[a] ed. Analysis of the metabolic profiles of the mutant strains showed the complete collapse of DDMM biosynthesis, but upon overexpression of the SARP regulatory gene msnR1 in each mutant new intermediates were detected. The compounds were isolated, and their structures were elucidated. Based on the results the specific functions of several enzymes were determined, and a pathway for mensacarcin biosynthesis is proposed.
The biologically active compound mensacarcin is produced by Streptomyces bottropensis. The cosmid cos2 contains a large part of the mensacarcin biosynthesis gene cluster. Heterologous expression of this cosmid in Streptomyces albus J1074 led to the production of the intermediate didesmethylmensacarcin (DDMM). In order to gain more insights into the biosynthesis, gene inactivation experiments were carried out by l-Red/ETmediated recombination, and the deletion mutants were introduced into the host S. albus. In total, 23 genes were inactivat-
Introduction Streptomyces bottropensis (formerly Streptomyces sp. Gç C4/4) was found in a soil sample collected at the University of Gçttingen in 1998.[1] Analysis of the culture broth of this strain revealed the production of new polyketides, namely mensacarcin (1, Scheme 1), which is a potential anti-tumor drug, and rishirilides A and B, which act as a2-macroglobulin inhibitors.[2] Mensacarcin is a hexahydroanthracene with nine stereogenic centers, and its structure shows high similarity to that of the anticancer compound cervicarcin, which is produced by Streptomyces ogaensis.[3] The high degree of oxygenation and particularly the two epoxy moieties (one in the side chain and one in the core structure) in mensacarcin result in a pharmaceutically interesting compound. The epoxy moiety in the side chain of mensacarcin is mainly responsible for the anti-tumor activity; hydroxylation of the C ring can modulate its activity.[1] By itself, mensacarcin has a mean cytostatic activity of 1.3 mm against 60 cell lines, with the best results for CCRF-CEM and NCI-
H322M, and is even active against the multidrug-resistant cellline Kato III.[4] Further studies with mensacarcin derivatives with modified side chains led to the hypothesis that mensacarcin might act by DNA alkylation, but the details at the molecular level are unclear.[4] The elucidation of the biosynthesis of mensacarcin and other natural compounds is of great interest, because the genes from different biosynthesis pathways can be combined to generate new biologically active compounds with improved pharmacological effects. Therefore, a detailed understanding of the function and the correct order of the individual enzymes is required. We previously cloned the biosynthesis gene cluster of mensacarcin (Figure 1).[5] Sequence analysis of the PKSII cluster revealed 34 open reading frames (ORFs) comprising 23 structural genes, two regulatory genes, and nine genes encoding hypothetical proteins with unknown function. When cos2, which contains most genes of the cluster but lacks genes involved in the incorporation of methyl groups at C9 and C10, was expressed in Streptomyces albus J1074, didesmethylmensacarcin (DDMM, 2; Scheme 1) was produced. In addition to the minimal PKS genes (msnK1, msnK2, msnK3), three cyclase genes (msnC1, msnC2, msnC3), three ketoreductase genes (msnO1, msnO10, msnO11), two oxidoreductase genes (msnO3, msnO9), one thioesterase gene (msnH7), and one dehydrogenase gene (msnH6), the biosynthetic gene cluster contains three luciferase-like monooxygenase genes (msnO2, msnO4, msnO8) and three anthron oxygenase genes (msnO5, msnO6, msnO7). As the specific function of most of these genes cannot be predicted by comparing the deduced amino acid sequences with known proteins, gene inactivation experiments were carried out.
[a] S. Maier,+ T. Heitzler,+ Dr. K. Asmus, Dr. U. Hardter, K. Hesselbach, Prof. Dr. A. Bechthold Institut fr Pharmazeutische Biologie und Biotechnologie Albert-Ludwigs Universitt Stefan-Meier-Strasse 19, 79104 Freiburg (Germany) E-mail: [email protected] [b] Dr. E. Brçtz, Dr. T. Paululat Organic Chemsitry II, Universitt Siegen Adolf-Reichwein-Strasse 2, 57068 Siegen (Germany) [c] Dr. E. Brçtz Present address: Helmholtz Institut fr Pharmazeutische Forschung Saarland Postfach 151150, 66041 Saarbrcken (Germany) [+] These authors contributed equally to this work. Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cbic.201500048.
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Full Papers lation of intermediates influences the biosynthesis of mensacarcin by a negative feedback mechanism. Notably, the mutants described here were all complemented successfully by expressing the replaced gene in trans, thus excluding the possibility of polar effects on the expression of genes located downstream. Deletion of msnH0–msnH4 did not affect DDMM production In order to verify that genes msnH0, msnT1, msnT2, msnH1, msnH2, msnH3, and msnH4 (all upstream of msnO1; left of the cluster in Figure 1) are not inScheme 1. Mensacarcin (1), didesmethylmensacarcin (DDMM, 2), MsnKH1 (SEK43, 3), MsnSM2 (4), MsnKP2 (5), volved in mensacarcin biosynMsnKP2a (6), MsnSM3 (7), MsnSM4 (8), and MsnKP1 (9). Compounds 3, 4, 7, 8, and 9 were isolated and characterthesis, gene inactivation experiized in this study. ments were performed. DDMM production was not affected by inactivation of msnH0, msnH4, or all seven genes (Table 1).
Results and Discussion
High production of didesmethylmensacarcin and intermediates of the biosynthesis is dependent on overexpression of msnR1
Deletion of msnH7 results in decreased DDMM production, and deletion of msnH6 results in abrogation MsnH7 is similar to thioesterases, which are not often part of the polyketide biosynthesis machinery.[7, 8] msnH7 was inactivated on cos2, and the mutated cosmid was introduced together with pKR1 into S. albus. Analysis of the production culture indicated decreased DDMM production. In 2011 Whicher et al. described inactivation of redJ (encoding a thioesterase involved in prodiginine biosynthesis). This did not result in a breakdown of the biosynthesis. It was shown that RedJ is involved in discharging dodecanoic acid from the acyl-carrier protein.[9] Thus, we assume that MsnH7 has a similar function in mensacarcin biosynthesis. MsnH6 belongs to the short-chain dehydrogenases/reductases (SDR) superfamily.[10] The SDR family contains a large number of functionally heterogeneous proteins with low sequence identities in pair-wise comparisons (about 15–30 %). Most SDR enzymes have an N- or C-terminal transmembrane domain; they can exist as discrete peptides but quite often form part of multienzyme complexes. The SDR superfamily is divided into five families on the basis of sequence motif: divergent, classical, intermediate, extended, and complex SDRs.[10] Complex SDRs are NADP(H)-binding proteins in which the SDR region catalyzes b-ketoacyl reductions.[10] In order to investigate the function of MsnH6, msnH6 was deleted on cos2. The mutated cos2 was introduced together with pKR1 into S. albus. DDMM production was repressed (as well as the formation of intermediates), thus indicating an essential function for MsnH6 in mensacarcin biosynthesis. As described for other enzymes (e.g., ref. [11]), MsnH6 seems to be part of the PKS complex.
Cosmid cos2, which contains a large part of the mensacarcin biosynthesis gene cluster, was introduced into S. albus J1074 by intergeneric conjugation. Cultivation of this strain led to the production of DDMM.[5] In order to analyze the functions of the several genes of the cluster, they were inactivated on cos2 by l-Red/ET-mediated recombination. The resulting mutant cosmids were introduced into S. albus and the strain was cultivated in DNPM medium (soytone (7.5 g L 1), baker’s yeast 3-(N-morpholino)propanesulfonic acid (MOPS; (5 g L 1), 21 g L 1, pH 6.8)). At various time points, the supernatant and the cells were extracted with ethyl acetate, and the metabolic profiles were analyzed by HPLC/ESI-MS at 254 nm. Surprisingly, expression of cosmids containing deletions in msnO4, msnO5, msnO6, msnO7, msnO8, or msnO9 resulted in no compound. In addition, deletion of msnR1 (SARP transcription regulator gene) also led to a breakdown of the whole DDMM biosynthesis. When plasmid pKR1 (containing msnR1 transcribed from the ermE-up promoter) was introduced into S. albus containing cos2, DDMM production increased by a factor of four (data not shown).[4] Consequently, pKR1 was also introduced into S. albus containing the deletion cosmids. Interestingly, overexpression of msnR1 led to the accumulation of novel products at 1– 2 mg L 1. Similar results were described by Chen et al:[6] they were able to increase the production of intermediates of fredericamycin biosynthesis 12-fold by overexpression of the pathway-specific activator fdmR1.[6] We postulated that the accumu-
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Figure 1. Genetic organization of the mensacarcin biosynthetic gene cluster and cos2 (containing the didesmethylmensacarcin biosynthetic gene cluster). Genes encoding hypothetical proteins are shown in white; two genes encoding transposases are in light gray; regulatory genes are in dark gray; three genes encoding the three non-contiguous genes minimal PKS are in black; genes encoding oxygenases are striped; genes encoding cyclases are spotted; genes for SAM regeneration/ methylation are and chequered.
actinorhodin cluster.[13–15] ActKR, a C9-ketoreductase, has two important functions: cyclization of the first ring (between C7 C12) and regiospecific reduction of the C9 carbonyl group to a hydroxyl group. The mensacarcin cluster contains three ketoreductase genes: msnO1, msnO10, and msnO11.[5] MsnO1 and MsnO11 show high similarity to BenL (C19-ketoreductase) from the benastatin pathway[16] and to ActIII (C9-ketoreductase) from the actinorhodin pathway, respectively.[17] MsnO10 did not show significant similarity to either BenL or ActIII. Lackner et al. showed that the cyclisation pattern as well as the regioselectivity of a ketoreduction can be predicted from sequence alignment.[18] It was shown that ketoreductases can be divided into four types on the basis of regioselectivity and cyclisation pattern: clade I (C19-ketoreductases responsible for the reduction of pentangular polyketides), clade II (C17-ketoreductases from the anthracycline biosynthesis), clade III (C15-ketoreductases from the biosynthesis of angucycline antibiotics), and clade IV (specific C9-ketoreductases from the biosynthesis of benzoisochromane quinines such as actinorhodin and granaticin, anthracyclines such as aclacinomycin, and angucyclines such as jadomycin and urdamycin). Clades I–III form a superfamily that reduces a carbonyl group in either terminal or ring positions. Based on sequence similarity (data not shown). MsnO11 belongs to clade IV, whereas MsnO1 and MsnO10 are members of clade II. To investigate the functions of the ketoreductases of the mensacarcin cluster, gene inactivation experiments were performed. Analysis of the metabolic profiles of the resulting strains revealed the production of new compounds. S. albus containing cos2DmsnO1 and pKR1 produced MsnKH1 (3, m/z 367.2 [M H] ; Scheme 1). The structure of MsnKH1 is identical to that of SEK43.[19] SEK43 was described as a shunt product of the polyketide chain generated by the minimal PKS and a C9ketoreductase. Thus MsnO1 seems to act after C9-ketoreduction. Deletion of msnO11 (proposed C9-ketoreductase) led to complete loss of DDMM biosynthesis, and no shunt product
Table 1. Inactivation mutants of S. albus::cos2 and the produced intermediates. Inactivated gene(s) Description of mutant
Produced intermediate
msnH0 msnH1 msnH4 msnH0-H4 msnH6 msnH7 msnC1 msnC2 msnC3 msnO1 msnO2 msnO3
S. albus::cos2DO1/pKR1 S. albus::cos2DH1/pKR1 S. albus::cos2DH4/pKR1 S. albus::cos2DH0-H4/pKR1 S. albus::cos2DH6/pKR1 S. albus::cos2DH7/pKR1 S. albus::cos2DC1/pKR1 S. albus::cos2DC2/pKR1 S. albus::cos2DC3/pKR1 S. albus::cos2DO1/pKR1 S. albus::cos2DO2/pKR1 S. albus::cos2DO3/pKR1
msnO4 msnO5 msnO6 msnO7 msnO8 msnO9 msnO10 msnO11 msnR1
S. albus::cos2DO4/pKR1 S. albus::cos2DO5/pKR1 S. albus::cos2DO6/pKR1 S. albus::cos2DO7/pKR1 S. albus::cos2DO8/pKR1 S. albus::cos2DO9/pKR1 S. albus::cos2DO10/pKR1 S. albus::cos2DO11/pKR1 S. albus::cos2DR1
DDMM (2) DDMM (2) DDMM (2) DDMM (2) –[a] DDMM (2) –[a] –[a] –[a] MsnKH1 (3) DDMM (2), MsnSM3 (7) MsnSM3 (7), MsnSM4 (8), MsnSM5 MsnSM4 (8) MsnKP1 (9), MsnKP4 MsnKP1 (9), MsnKP4 DDMM (2) MsnKP2 (5), MsnKP2a (6) MsnKP1 (9) MsnSM1, MsnSM2 (4) –[a] –[a]
[a] Neither an intermediate nor a shunt product of DDMM was produced.
The ketoreductases MsnO1, MsnO10, and MsnO11 strongly control mensacarcin biosynthesis In PKS biosynthesis ketoreductases catalyze the reduction of a carbonyl group, most often of the C9 carbonyl, to a secondary alcohol. This reaction takes place after the formation of the first ring, typically between C7 C12 or C9 C14. Ketoreductases complement the work of the minimal PKS and promote the correct folding of the polyketide chain. Ketoreductases are also able to affect the orientation of the polyketide chain and thereby the cyclization pattern of the different secondary metabolites derived from PKSII metabolism. One of the best-studied ketoreductases is ActKR (also known as ActIII)[12] from the ChemBioChem 0000, 00, 0 – 0
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was observed. Complementation of the mutant strain with msnO11 recovered production, thus demonstrating that inactivation of msnO11 did not influence the expression of adjacent genes in the cluster. Thus MsnO11, like MsnH6, is essential for mensacarcin biosynthesis. In 2004, Hertweck et al. showed that the minimal PKS activity in enterocin biosynthesis depends on the activity of the ketoreductase EncD.[20] MsnO11 might play a similar role. Deletion of msnO10 led to the production of MsnSM2 (4, m/ z 432.1 [M H] ; Scheme 1). MsnSM2 is a new compound. The polyketide backbone consists of a cyclized heptaketide that carries a methyl group at C12 and a N-acetylcysteine residue at C3. Formation of N-acetylcysteine adducts have been described in the mycothiol-dependent detoxification pathway.[21] Compared to DDMM, MsnMS2 has a shorter chain-length and a different fold of the polyketide chain. The additional methyl group indicates a rearrangement of the polyketide chain during folding. MsnO10 is obviously needed for stabilizing the produced side chain, or even for stabilizing the entire PKS complex.
The luciferase-like monooxygenases MsnO2, MsnO4, and MsnO8 catalyze important tailoring steps in mensacarcin biosynthesis Flavin-dependent monooxygenases form a special group of tailoring enzymes. They catalyze the activation of molecular oxygen by a reduced flavin cofactor, thereby leading to incorporation of oxygen into a molecule.[31] Thus, flavoproteins are able to regio- and/or enantioselectively catalyze different reactions, such as hydroxylations, epoxidations, and Baeyer–Villiger oxidations.[32] Luciferases represent the best-studied group of flavoproteins. They are readily found in bioluminescence organisms such as glowworms, fungi, fish, and bacteria. Luciferins are the substrates for the bioluminescence reaction. Although luciferases catalyze a variety of chemical reactions, the only common factor is the need for oxygen.[33] The luciferase reaction is an oxidative process in which the substrate reaches an electronically excited condition. Upon radiating light, the excited-state intermediate reverts to the ground state.[34] The bacterial luciferase from Vibrio harveyi is a flavin-containing monooxygenase that oxidizes a long-chain aliphatic aldehyde to an aliphatic carboxylic acid while emitting visible light.[34] It forms a heterodimer (a and b subunits) to build a TIM barrel. The single active center is located on the a subunit.[35] The function of luciferase-like monooxygenases in the biosynthesis of streptomycete secondary metabolites has not been described. In the mensacarcin cluster three luciferase-like monooxygenase genes were identified (msnO2, msnO4 and msnO8). We recently showed that inactivation of msnO8 led to the formation of MsnKP2 (5, Scheme 1) and MsnKP2a (6).[4] msnO2 and msnO4 were deleted on cos2 in this study. Deletion of msnO2 led to the production of MsnSM3 (7, m/z 339.2 [M H] ; Scheme 1); deletion of msnO4 led to the production of MsnSM4 (8, m/z 530.1 [M+H] + and 552.1 [M+Na] + ). Surprisingly MsnSM4 retains the carboxyl group at C20, which is not present in MsnSM3. We assume that decarboxylation is somehow inhibited, maybe a consequence of the attached acetylcysteine residue, which is used for detoxification in actinomycetes.[36, 37] Thus, MsnKP2’ (5’; Scheme 2) and MsnSM4’ (8’) are most probably intermediates that become connected to the acetylcysteine residue. Based on the structures of the intermediates, MsnO2 is a dehydratase that introduces the double bond between C12 and C13 of the side
Not even shunt products are produced without cyclases MsnC1, MsnC2, and MsnC3 Ketoreductases, cyclases, and aromatases are needed in addition to the minimal PKS in order to form the backbone of an aromatic polyketide by PKSII. The highly reactive poly-b-intermediate produced by the minimal PKS can spontaneously cyclize. In order to prevent these inadvertent aldol reactions, catalyzing cyclases are needed.[22] Cyclases are cofactor-free enzymes that act in a chaperone-like manner. Although they form a heterogeneous group (in terms of sequence and protein structure), all catalyze specific aldol condensation reactions. Cyclases share the HxGTHxDxPxH motif, which is believed to be part of the active site.[23] Polyketide cyclization is often supported by aromatases, which catalyze the dehydration of cyclic alcohols thus leading to the formation of aromatic ring structures,[24] and by bifunctional cyclases, which can act as aromatases, methyltranferases, or ketoreductases.[23] Three cyclases (MsnC1, MsnC2, and MsnC3) are involved in mensacarcin biosynthesis.[5] Analysis of sequence homology revealed that MsnC1 and MsnC3 share the highest homology with ActVII (actinorhodin pathway) and aromatase ORF4 (granaticin pathway), both responsible for aromatization of the first ring.[12, 25] MsnC2 resembles ORF33 (granaticin biosynthesis)[26] and AlnR (alnumycin pathway), which catalyzes the second ring closure during polyketide biosynthesis.[27] MsnC1, msnC2 and msnC3 were inactivated on cos2, and the mutated cosmids were introduced with pKR1 into S. albus. As expected, no DDMM production was observed. Surprisingly, no shunt product or intermediate was detected. To exclude a systematic error by the inactivation method or effect by the inactivation on the transcription of other genes, each mutant was complemented with the intact cyclase gene; DDMM production was restored in all three cases. There are a number of reports that cyclases are essential for polyketide biosynthesis, but in most cases deletion of the cy-
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Scheme 2. Proposed pathway for mensacarcin biosynthesis showing involvement of mensacarcin enzymes.
chain of mensacarcin. MsnO4 might have multiple roles: introducing two hydroxy groups and one epoxy group into the third ring of mensacarcin. MsnO8 catalyzes epoxidation of the side chain.
sion, MsnO9 seems to be part of the PKS complex and influences the length of the polyketide chain. Three anthron oxygenases, MsnO5, MsnO6, and MsnO7 are involved in mensacarcin biosynthesis
MsnO9 is involved in determining the exact number of acetate units incorporated into the polyketo chain
Anthron oxygenases catalyze the oxidation of naphthacenone and anthron-type precursors of aromatic polyketides to the corresponding quinine derivatives. No cofactor is needed.[43] Anthron oxygenases have been termed “internal oxygenases”, as they use their substrates as reducing equivalents for the reduction of one oxygen to water.[23] A well-known anthron oxygenase is TcmH, which is involved in the conversion of the naphthacenone tetracenomycin F1 to the 5,12-naphthacenquinone tetracenomycin C3 in the biosynthesis of tetracenomycin. Other representatives are ActVA-Orf6 (actinorhodin pathway)[44] and DpgC (vancomycin pathway).[45] The anthron oxygenases in the DDMM pathway exhibit highest homology to AknX (aclacinomycin gene cluster).[46] By in vitro assays with emodineanthron as substrate, AknX was shown to result in the formation of emodineanthraquinone.[47] The genes encoding MsnO5, MsnO6, and MsnO7 were deleted on cos2. Expression of cosmids containing deletions of either msnO5 or msnO6 together with pKR1 resulted in the production of MsnKP1 (9, m/z 285.1 [M H] ; Scheme 1) and MsnKP4. Unfortunately, we were not able to isolate MsnKP4 for NMR studies. Cos2 containing a deleted msnO7 was still able to produce DDMM, however the production rate was considerably lower. As inactivation of msnO5 or msnO6 led to the production of shunt products (MsnKP1 and MsnKP4), this implies independent involvement of both enzymes in the reaction. Both seem to be responsible for the incorporation of oxygen at C5 and each seems to be able to catalyze the reaction. But both en-
BLASTp analysis revealed GrhO7 and ActVI-ORF2 as the closest characterized homologues of MsnO9. These proteins belong to the medium-chain reductases (MDR) superfamily and NADPH:quinone reductases/quinone oxidoreductase subfamily. Reactions catalyzed by NADPH:quinone reductases include the reduction of quinone to a semiquinone radical by transfer of an electron.[38] GrhO7 (griseorhodin biosynthetic gene cluster) was postulated to catalyze the formation of a double bond in dideoxygriseorhodin by dehydrogenation.[39] ActVI-ORF2 (actinorhodin pathway) is known to catalyze the stereospecific 1,4-reduction of (S)-DNPA (4-dihydro-9-hydroxy-1-methyl-10-oxo-3-Hnaphtho-[2,3-c]-pyran-3-(S)-acetic acid) to 5-DDHK (5-deoxy-dihydrokalafungin) at C15.[40] In order to investigate the role of MsnO9, the gene was deleted on cos2. Introduction of deleted cos2 together with pKR1 into S. albus led to the production of MsnKP1 (9, m/z 285.1 [M H] ; Scheme 1). MsnKP1 is identical to 2-hydroxyislandicin, which was first isolated from the plant Ventilago calyculata by Rao et al,[41] and lacks the side chain of mensacarcin (C15 instead of C19). It has been shown that the minimal PKS from the frenolicin pathway synthesizes polyketide chains of different lengths depending on the enzyme used for co-expression. When the aromatase/cyclase TcmN was used as a coexpression partner, nonaketides were produced. When a ketoreductase from the actinorhodin pathway was the co-expression partner, octa- and nonaketides were produced.[42] In concluChemBioChem 0000, 00, 0 – 0
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carcin, inactivation experiments were performed. Our results indicate that a surprisingly high number of enzymes is involved in determining the length and the folding of the elongated polyketide chain. Luciferase-like monooxygenases are involved in the later steps for the formation of epoxy moieties.
MsnO2, MsnO4, and MsnO8 need MsnO3 for activity
Experimental Section
Flavin-dependent oxidoreductases often need the reduced cofactor FMN for the transfer of oxygen. NAD(P)H flavin-dependent oxidoreductases catalyze the reduction of FMN by NAD(P)H. The transfer of reduced FMN to the oxidoreductase can occur by diffusion or directly by protein–protein interaction. An example of the diffusion process is the two-component enzyme system LuxG/LuxAB from Photobacterium leiognathi. LuxAB, the oxidoreductase, receives reduced FMN from the NADH:FMN oxidoreductase LuxG. Because of the instability of reduced FMN, it was postulated that the affinity to LuxAB is quite high and the binding appears very quick to minimize FMN oxidation and formation of H2O2.[48] Protein–protein transfer interactions have been postulated for the systems in Vibrio fischeri and V. harveyi.[49] We recently showed that MsnO8 activity is dependent on the activity of the NADH-dependent flavin reductase MsnO3.[4] In the present study, expression of cos2 containing deleted msnO3 led to the production of a marginal amount of DDMM as well as MsnSM3 (7, Scheme 1), MsnSM4 (8), and numerous of degradation or shunt products. This indicates that MsnO3 acts in a two-component system to provide reduced FMN to MsnO2, MsnO4, and MsnO8.
l-Red/ET-mediated recombination: Inactivation of genes on the cosmid cos2 was carried out by l-Red/ET-mediated recombination as previously described.[50] The targeted gene was replaced with a spectinomycin-resistance cassette, which was subsequently removed by restriction digestion with NdeI followed by ligation. Insertion and removal of the spectinomycin cassette and intergeneric conjugation were as previously described.[4] Insertion of the spectinomycin cassette and inactivation of the gene were verified by PCR (primer sequences for the amplification of the spectinomycin cassettes and verification are in the Supporting Information). Cloning of genes for complementation: For generating the complementation plasmids of msnO1, msnO4–msnO11 and msnR1, pUWL-H-tnp[51, 52] was used as the expression vector. pUWL-H-tnp was linearized by restriction digestion with BamHI and HindIII. All genes were amplified by PCR from cos2,[53] with forward and reverse primers (sequences given in the Supporting Information) containing HindIII and BamHI restriction sites. The PCR products were restriction digested and ligated into the linearized pUWL-Htnp. After successful insertion, the constructed plasmid was digested with BamHI and SpeI. MsnR1 was amplified by PCR with primers F-SARP and R-SARP (incorporating BglII and SpeI restriction sites). The ligation of this PCR product into the previously constructed plasmids resulted in plasmids pKO4R1–pKO11R1. For the construction of pKO2R1, pKRO3R1, pKRC1R1–pKRC3R1 and pKH6R1, the first step was to construct pKR1 from pUWL-H-tnp. For this, msnR1 was amplified from cos2 with primers pKR1-F and pKR1-R and cloned at HindIII and SpeI sites into pUWL-H-tnp. msnO2, msnO3, msnC1-C3, and msnH6 were amplified by PCR from cos2 and digested (restriction sites ClaI and HindIII in the primers). pKR1 was similarly digested and ligated to the PCR products. As msnH7 is directly upstream of msnR1 in the msn cluster, these genes were amplified from cos2 as one PCR product (primers KR1-F and KH7-R). This and pUWL-H-tnp were each cut with HindIII and SpeI, and ligated to obtain pKRH7R1.
Proposed biosynthesis pathway of mensacarcin Mensacarcin (1) is constructed from 10 acetate units. An enzyme complex consisting of the minimal PKS (MsnK1, MsnK2, and MsnK3), the C9-ketoreductase (MsnO11), three cyclases (MsnC1, MsnC2, and MsnC3), and a short-chain dehydrogenase (MsnH6) is required for the formation of a polyketo derivative. In addition, two ketoreductases (MsnO1 and MsnO11) and the medium-chain instead of dehydrogenase/reductase (MsnO9) are involved in determining the exact number of acetate units and the folding of the polyketide chain. The anthron oxygenases (MsnO5 and MsnO6, but not MsnO7) also have an impact on the chain length of the polyketide chain; the thioesterase (MsnH7) catalyzes the release of the polyketide from the ACP. The first correctly folded intermediate containing all carbons is MsnSM3 (7). MsnO2 converts it into MsnSM4’ (8’), which is converted into MsnKP2’ (5’) by MsnO4. Finally, the introduction of the epoxy group of the side chain is catalyzed by MsnO8. MsnO2, msnO4, and MsnO8 require the activity of MsnO3 for full activity (Scheme 2).
Production and analysis of didesmethylmensacarcin and intermediates: The cosmids and plasmids were transferred by intergeneric conjugation into S. albus strain J1074.[54] The exconjugants were cultured for 24 h in tryptic soy broth (TSB; 30 g L 1) at 28 8C with agitation. This pre-culture was used to inoculate DNPM production medium (soytone (7.5 g L 1), baker’s yeast (5 g L 1), 3-(Nmorpholino)propanesulfonic acid (MOPS, 21 g L 1), pH 6.8). After 96–120 h incubation (28 8C, 180 rpm), the cells were removed by centrifugation, then the medium was adjusted to pH 4 and extracted twice with an equal volume of ethyl acetate. The organic extracts were dried in vacuum and dissolved in methanol for analysis on an 1100 series LC/MS system (254 nm; Agilent Technologies) with electrospray ionization (ESI). The LC system was equipped with a XBridge C-18 column (3.5 mm particle size, 3.9 20 mm; Waters) followed by a second XBridge C-18 column (3.5 mm particle size, 4.6 100 mm). The acetonitrile gradient was as previously described for mensacarcin analysis.[5]
Conclusion Mensacarcin is a potent anticancer agent.[1] It is highly decorated with oxygen and even contains two epoxy moieties. In order to get a deeper insight into the biosynthesis of mensa-
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Purification of MsnKP1: To purify MsnKP1, the supernatant of S. albus::cos2DO9/pKR1 (4 L) was extracted with ethyl acetate. Prepara-
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tive TLC followed by preparative HPLC led to 6.6 mg of pure MsnKP1. The dried extract was dissolved in methanol and applied to a silica gel TLC plate (ADAMANT silica gel 60 F254 ; Macherey– Nagel). The mobile phase was acetic acid (0.05 %) in CH2Cl2/methanol (9:1, v/v). For preparative HPLC an Zorbax-C18-system (Agilent Technologies) was equipped with two columns (5 mm particle size, 50 9.4 mm followed by 5 mm particle size, 150 9.4 mm). The eluent was monitored at 254 and 325 nm with a diode array detector (solvent A: acetonitrile with acetic acid (0.5 % v/v), solvent B: acetic acid (0.5 % v/v); gradient: 40 % A (4 min), linear gradient to 95 % A (11 min), 95 % A (4 min), linear gradient to 40 % A (4 min); flow-rate 2 mL min 1).
Financial support for this research was provided by the RTG 1976 (cofactor dependent enzymes) funded by the Deutsche Forschungsgemeinschaft (DFG). Keywords: biosynthesis · didesoxymensacarcin · mensacarcin · polyketides · structure elucidation [1] L. F. Tietze, K. M. Gericke, I. Schuberth, Eur. J. Org. Chem. 2007, 4563 – 4577. [2] L. F. Tietze, S. G. Stewart, M. E. Polomska, A. Modi, A. Zeeck, Chem. Eur. J. 2004, 10, 5233 – 5242. [3] J. Nagatsu, S. Suzuki, J. Antibiot. 1963, 16, 203 – 206. [4] S. Maier, T. Pflger, S. Loesgen, K. Asmus, E. Brçtz, T. Paululat, A. Zeeck, S. Andrade, A. Bechthold, ChemBioChem 2014, 15, 749 – 756. [5] X. Yan, K. Probst, A. Linnenbrink, M. Arnold, T. Paululat, A. Zeeck, A. Bechthold, ChemBioChem 2012, 13, 224 – 230. [6] Y. Chen, E. Wendt-Pienkoski, S. R. Rajski, B. Shen, J. Biol. Chem. 2009, 284, 24735 – 24743. [7] J. A. Kalaitzis, Q. Cheng, D. Meluzzi, L. Xiang, M. Izumikawa, P. C. Dorrestein, B. S. Moore, Bioorg. Med. Chem. 2011, 19, 6633 – 6638. [8] Y. Tang, A. T. Koppisch, C. Khosla, Biochemistry 2004, 43, 9546 – 9555. [9] J. R. Whicher, G. Florova, P. K. Sydor, R. Singh, M. Alhamadsheh, G. L. Challis, K. A. Reynolds, J. L. Smith, J. Biol. Chem. 2011, 286, 22558 – 22569. [10] Y. Kallberg, U. Oppermann, H. Jçrnvall, B. Persson, Eur. J. Biochem. 2002, 269, 4409 – 4417. [11] J. Zhan, K. Watanabe, Y. Tang, ChemBioChem 2008, 9, 1710 – 1715. [12] R. McDaniel, S. Ebert-Khosla, D. A. Hopwood, C. Khosla, J. Am. Chem. Soc. 1994, 116, 10855 – 10859. [13] J. A. Kalaitzis, B. S. Moore, J. Nat. Prod. 2004, 67, 1419 – 1422. [14] P. Javidpour, T. P. Korman, G. Shakya, S.-C. Tsai, Biochemistry 2011, 50, 4638 – 4649. [15] P. Javidpour, J. Bruegger, S. Srithahan, T. P. Korman, M. P. Crump, J. Crosby, M. D. Burkart, S.-C. Tsai, Chem. Biol. 2013, 20, 1225 – 1234. [16] Z. Xu, A. Schenk, C. Hertweck, J. Am. Chem. Soc. 2007, 129, 6022 – 6030. [17] A. T. Hadfield, C. Limpkin, W. Teartasin, T. J. Simpson, J. Crosby, M. P. Crump, Structure 2004, 12, 1865 – 1875. [18] G. Lackner, A. Schenk, Z. Xu, K. Reinhardt, Z. S. Yunt, J. Piel, C. Hertweck, J. Am. Chem. Soc. 2007, 129, 9306 – 9312. [19] R. McDaniel, S. Ebert-Khosla, D. A. Hopwood, C. Khosla, Nature 1995, 375, 549 – 554. [20] C. Hertweck, L. Xiang, J. A. Kalaitzis, Q. Cheng, M. Palzer, B. S. Moore, Chem. Biol. 2004, 11, 461 – 468. [21] G. L. Newton, R. C. Fahey, Arch. Microbiol. 2002, 178, 388 – 394. [22] H. Zhou, Y. Li, Y. Tang, Nat. Prod. Rep. 2010, 27, 839 – 868. [23] C. Hertweck, A. Luzhetskyy, Y. Rebets, A. Bechthold, Nat. Prod. Rep. 2007, 24, 162 – 190. [24] C. Hertweck, Angew. Chem. Int. Ed. 2009, 48, 4688 – 4716; Angew. Chem. 2009, 121, 4782 – 4811. [25] T. W. Yu, M. J. Bibb, W. P. Revill, D. A. Hopwood, J. Bacteriol. 1994, 176, 2627 – 2634. [26] M.-R. Deng, J. Guo, X. Li, C.-H. Zhu, H.-H. Zhu, Antonie Van Leeuwenhoek 2011, 100, 607 – 617. [27] T. Oja, K. Palmu, H. Lehmussola, O. Leppranta, K. Hnnikinen, J. Niemi, P. Mntsl, M. Mets-Ketel, Chem. Biol. 2008, 15, 1046 – 1057. [28] K. Fritzsche, K. Ishida, C. Hertweck, V. Uni, J. Am. Chem. Soc. 2008, 130, 8307 – 8316. [29] B. Ostash, Y. Rebets, V. Yuskevich, A. Luzhetskyy, V. Tkachenko, V. Fedorenko, Folia Microbiol. 2003, 48, 484 – 488. [30] J. Staunton, K. J. Weissman, Nat. Prod. Rep. 2001, 18, 380 – 416. [31] V. Massey, J. Biol. Chem. 1994, 269, 22459 – 22462. [32] W. J. H. van Berkel, N. M. Kamerbeek, M. W. Fraaije, J. Biotechnol. 2006, 124, 670 – 689. [33] R. Boyle, Philos. Trans. R. Soc. Lond. Biol. Sci. 1668, 2, 581 – 600. [34] A. J. Fisher, T. B. Thompson, J. B. Thoden, T. O. Baldwin, I. Rayment, J. Biol. Chem. 1996, 271, 21956 – 21968.
Purification of MsnSM3 and MsnSM4: To purify MsnSM3 and MsnSM4, the supernatant of S. albus::cos2DO4/pKR1 (5 L) was extracted with ethyl acetate. Solid-phase extraction followed by preparative TLC, preparative HPLC, and gel filtration led to 5.5 mg of pure MsnSM3 and 1.5 mg of MsnSM4. The crude extract was fractionated in an Oasis HLB20 35 cc extraction cartridge (Waters) with a methanol/water gradient. Separation by TLC (silica gel 60 F254 ; Machery–Nagel) was performed with acetic acid (0.1 %) in CH2Cl2/ methanol (1:9, v/v). Preparative HPLC was carried out with a system of two Xbridge C18-columns (3.5 mm particle size, 20 4.6 mm followed by 3.5 mm particle size, 100 4.6 mm). The eluent was monitored at 254 nm using a diode array detector (40 % A (4 min), linear gradients to 45 % A (4 min), to 95 % A (10 min), 95 % A (2 min), wash with 40 % A (4 min); flow-rate 0.5 mL min 1. Finally, MsnSM4 was purified by gel filtration on a Sephadex LH20 column (GE Healthcare) with methanol. Purification of MsnSM2: The supernatant of S. albus::cos2DO10/ pKR1 (4 L) was extracted with ethyl acetate. Solid-phase extraction followed by preparative TLC and gel filtration led to pure MsnSM2. The crude extract was fractionated on an Oasis HLB20 35 cc extraction cartridge (Waters) with a methanol/water gradient. Separation by TLC (silica gel 60 F254 ; Machery–Nagel) was with acetic acid (0.5 %) in CH2Cl2/methanol (9:1, v/v). Finally, MsnSM2 was purified by gel filtration on a Sephadex LH20 column with methanol, thereby resulting in 1 mg of MsnSM2. Purification of MsnKH1: The supernatant of S. albus::cos2DO11/ pKR1 (5 L) was extracted with ethyl acetate. Solid-phase extraction followed by preparative TLC, preparative HPLC and gel filtration led to pure MsnKH1. The crude extract was fractionated on an Oasis HLB20 35 cc extraction cartridge with a methanol/water gradient. Separation by TLC (silica gel 60 F254 ; Macherey–Nagel) was with acetic acid (0.5 %) in acetonitrile/water (85:10, v/v). Preparative HPLC was carried out with a system of two Xbridge C18-columns (3.5 mm particle size, 20 3.9 mm followed by 3.5 mm particle size, 100 4.6 mm). The eluent was monitored at 254 nm with a diode array detector (30 % A, linear gradients to 70 % A (8 min), then to 95 % A (1 min), 95 % A (3 min), wash with 30 % A (5 min); flow-rate 0.5 mL min 1). Finally, MsnKH1 was purified by gel filtration on a Sephadex LH20 column with methanol, thereby resulting in 5.1 mg of MsnKH1. Structure elucidation: NMR spectra were measured on a VNMR-S 600 spectrometer (1H: 600 MHz, 13C: 150 MHz; Varian) at 35 8C ([D6]DMSO) or 25 8C (other solvents). Residual solvent signals were used as internal references ([D6]DMSO: dH = 2.50 ppm, dC = 39.5 ppm; CD3OD: dH = 3.30 ppm, dC = 49.0 ppm). HR-ESI-MS were recorded on an LTQ Orbitrap XL (Thermo Scientific) and an Apex-Q III (Bruker). MsnSM4 was analyzed on a model 1100 HPLC/ESI-MS device (Agilent Technologies) coupled to a MSD.
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FULL PAPERS MsnO2, MsnO4, and MsnO8: We elucidated the components of the Streptomyces bottropensis mensacarcin biosynthesis gene cluster by selective deletion and complementation in a heterologous expression system. Three luciferase-like monooxygenases are involved in later steps of mensacarcin biosynthesis.
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S. Maier, T. Heitzler, K. Asmus, E. Brçtz, U. Hardter, K. Hesselbach, T. Paululat, A. Bechthold* && – && Functional Characterization of Different ORFs Including LuciferaseLike Monooxygenase Genes from the Mensacarcin Gene Cluster
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Functional Characterization of Different ORFs Including Luciferase-Like Monooxygenase Genes from the Mensacarcin Gene Cluster Sarah Maier,[a] Tanja Heitzler,[a] Katharina Asmus,[a] Elke Brçtz,[b, c] Uwe Hardter,[a] Katharina Hesselbach,[a] Thomas Paululat,[b] and Andreas Bechthold*[a] ed. Analysis of the metabolic profiles of the mutant strains showed the complete collapse of DDMM biosynthesis, but upon overexpression of the SARP regulatory gene msnR1 in each mutant new intermediates were detected. The compounds were isolated, and their structures were elucidated. Based on the results the specific functions of several enzymes were determined, and a pathway for mensacarcin biosynthesis is proposed.
The biologically active compound mensacarcin is produced by Streptomyces bottropensis. The cosmid cos2 contains a large part of the mensacarcin biosynthesis gene cluster. Heterologous expression of this cosmid in Streptomyces albus J1074 led to the production of the intermediate didesmethylmensacarcin (DDMM). In order to gain more insights into the biosynthesis, gene inactivation experiments were carried out by l-Red/ETmediated recombination, and the deletion mutants were introduced into the host S. albus. In total, 23 genes were inactivat-
Introduction Streptomyces bottropensis (formerly Streptomyces sp. Gç C4/4) was found in a soil sample collected at the University of Gçttingen in 1998.[1] Analysis of the culture broth of this strain revealed the production of new polyketides, namely mensacarcin (1, Scheme 1), which is a potential anti-tumor drug, and rishirilides A and B, which act as a2-macroglobulin inhibitors.[2] Mensacarcin is a hexahydroanthracene with nine stereogenic centers, and its structure shows high similarity to that of the anticancer compound cervicarcin, which is produced by Streptomyces ogaensis.[3] The high degree of oxygenation and particularly the two epoxy moieties (one in the side chain and one in the core structure) in mensacarcin result in a pharmaceutically interesting compound. The epoxy moiety in the side chain of mensacarcin is mainly responsible for the anti-tumor activity; hydroxylation of the C ring can modulate its activity.[1] By itself, mensacarcin has a mean cytostatic activity of 1.3 mm against 60 cell lines, with the best results for CCRF-CEM and NCI-
H322M, and is even active against the multidrug-resistant cellline Kato III.[4] Further studies with mensacarcin derivatives with modified side chains led to the hypothesis that mensacarcin might act by DNA alkylation, but the details at the molecular level are unclear.[4] The elucidation of the biosynthesis of mensacarcin and other natural compounds is of great interest, because the genes from different biosynthesis pathways can be combined to generate new biologically active compounds with improved pharmacological effects. Therefore, a detailed understanding of the function and the correct order of the individual enzymes is required. We previously cloned the biosynthesis gene cluster of mensacarcin (Figure 1).[5] Sequence analysis of the PKSII cluster revealed 34 open reading frames (ORFs) comprising 23 structural genes, two regulatory genes, and nine genes encoding hypothetical proteins with unknown function. When cos2, which contains most genes of the cluster but lacks genes involved in the incorporation of methyl groups at C9 and C10, was expressed in Streptomyces albus J1074, didesmethylmensacarcin (DDMM, 2; Scheme 1) was produced. In addition to the minimal PKS genes (msnK1, msnK2, msnK3), three cyclase genes (msnC1, msnC2, msnC3), three ketoreductase genes (msnO1, msnO10, msnO11), two oxidoreductase genes (msnO3, msnO9), one thioesterase gene (msnH7), and one dehydrogenase gene (msnH6), the biosynthetic gene cluster contains three luciferase-like monooxygenase genes (msnO2, msnO4, msnO8) and three anthron oxygenase genes (msnO5, msnO6, msnO7). As the specific function of most of these genes cannot be predicted by comparing the deduced amino acid sequences with known proteins, gene inactivation experiments were carried out.
[a] S. Maier,+ T. Heitzler,+ Dr. K. Asmus, Dr. U. Hardter, K. Hesselbach, Prof. Dr. A. Bechthold Institut fr Pharmazeutische Biologie und Biotechnologie Albert-Ludwigs Universitt Stefan-Meier-Strasse 19, 79104 Freiburg (Germany) E-mail: [email protected] [b] Dr. E. Brçtz, Dr. T. Paululat Organic Chemsitry II, Universitt Siegen Adolf-Reichwein-Strasse 2, 57068 Siegen (Germany) [c] Dr. E. Brçtz Present address: Helmholtz Institut fr Pharmazeutische Forschung Saarland Postfach 151150, 66041 Saarbrcken (Germany) [+] These authors contributed equally to this work. Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cbic.201500048.
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Full Papers lation of intermediates influences the biosynthesis of mensacarcin by a negative feedback mechanism. Notably, the mutants described here were all complemented successfully by expressing the replaced gene in trans, thus excluding the possibility of polar effects on the expression of genes located downstream. Deletion of msnH0–msnH4 did not affect DDMM production In order to verify that genes msnH0, msnT1, msnT2, msnH1, msnH2, msnH3, and msnH4 (all upstream of msnO1; left of the cluster in Figure 1) are not inScheme 1. Mensacarcin (1), didesmethylmensacarcin (DDMM, 2), MsnKH1 (SEK43, 3), MsnSM2 (4), MsnKP2 (5), volved in mensacarcin biosynMsnKP2a (6), MsnSM3 (7), MsnSM4 (8), and MsnKP1 (9). Compounds 3, 4, 7, 8, and 9 were isolated and characterthesis, gene inactivation experiized in this study. ments were performed. DDMM production was not affected by inactivation of msnH0, msnH4, or all seven genes (Table 1).
Results and Discussion
High production of didesmethylmensacarcin and intermediates of the biosynthesis is dependent on overexpression of msnR1
Deletion of msnH7 results in decreased DDMM production, and deletion of msnH6 results in abrogation MsnH7 is similar to thioesterases, which are not often part of the polyketide biosynthesis machinery.[7, 8] msnH7 was inactivated on cos2, and the mutated cosmid was introduced together with pKR1 into S. albus. Analysis of the production culture indicated decreased DDMM production. In 2011 Whicher et al. described inactivation of redJ (encoding a thioesterase involved in prodiginine biosynthesis). This did not result in a breakdown of the biosynthesis. It was shown that RedJ is involved in discharging dodecanoic acid from the acyl-carrier protein.[9] Thus, we assume that MsnH7 has a similar function in mensacarcin biosynthesis. MsnH6 belongs to the short-chain dehydrogenases/reductases (SDR) superfamily.[10] The SDR family contains a large number of functionally heterogeneous proteins with low sequence identities in pair-wise comparisons (about 15–30 %). Most SDR enzymes have an N- or C-terminal transmembrane domain; they can exist as discrete peptides but quite often form part of multienzyme complexes. The SDR superfamily is divided into five families on the basis of sequence motif: divergent, classical, intermediate, extended, and complex SDRs.[10] Complex SDRs are NADP(H)-binding proteins in which the SDR region catalyzes b-ketoacyl reductions.[10] In order to investigate the function of MsnH6, msnH6 was deleted on cos2. The mutated cos2 was introduced together with pKR1 into S. albus. DDMM production was repressed (as well as the formation of intermediates), thus indicating an essential function for MsnH6 in mensacarcin biosynthesis. As described for other enzymes (e.g., ref. [11]), MsnH6 seems to be part of the PKS complex.
Cosmid cos2, which contains a large part of the mensacarcin biosynthesis gene cluster, was introduced into S. albus J1074 by intergeneric conjugation. Cultivation of this strain led to the production of DDMM.[5] In order to analyze the functions of the several genes of the cluster, they were inactivated on cos2 by l-Red/ET-mediated recombination. The resulting mutant cosmids were introduced into S. albus and the strain was cultivated in DNPM medium (soytone (7.5 g L 1), baker’s yeast 3-(N-morpholino)propanesulfonic acid (MOPS; (5 g L 1), 21 g L 1, pH 6.8)). At various time points, the supernatant and the cells were extracted with ethyl acetate, and the metabolic profiles were analyzed by HPLC/ESI-MS at 254 nm. Surprisingly, expression of cosmids containing deletions in msnO4, msnO5, msnO6, msnO7, msnO8, or msnO9 resulted in no compound. In addition, deletion of msnR1 (SARP transcription regulator gene) also led to a breakdown of the whole DDMM biosynthesis. When plasmid pKR1 (containing msnR1 transcribed from the ermE-up promoter) was introduced into S. albus containing cos2, DDMM production increased by a factor of four (data not shown).[4] Consequently, pKR1 was also introduced into S. albus containing the deletion cosmids. Interestingly, overexpression of msnR1 led to the accumulation of novel products at 1– 2 mg L 1. Similar results were described by Chen et al:[6] they were able to increase the production of intermediates of fredericamycin biosynthesis 12-fold by overexpression of the pathway-specific activator fdmR1.[6] We postulated that the accumu-
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Figure 1. Genetic organization of the mensacarcin biosynthetic gene cluster and cos2 (containing the didesmethylmensacarcin biosynthetic gene cluster). Genes encoding hypothetical proteins are shown in white; two genes encoding transposases are in light gray; regulatory genes are in dark gray; three genes encoding the three non-contiguous genes minimal PKS are in black; genes encoding oxygenases are striped; genes encoding cyclases are spotted; genes for SAM regeneration/ methylation are and chequered.
actinorhodin cluster.[13–15] ActKR, a C9-ketoreductase, has two important functions: cyclization of the first ring (between C7 C12) and regiospecific reduction of the C9 carbonyl group to a hydroxyl group. The mensacarcin cluster contains three ketoreductase genes: msnO1, msnO10, and msnO11.[5] MsnO1 and MsnO11 show high similarity to BenL (C19-ketoreductase) from the benastatin pathway[16] and to ActIII (C9-ketoreductase) from the actinorhodin pathway, respectively.[17] MsnO10 did not show significant similarity to either BenL or ActIII. Lackner et al. showed that the cyclisation pattern as well as the regioselectivity of a ketoreduction can be predicted from sequence alignment.[18] It was shown that ketoreductases can be divided into four types on the basis of regioselectivity and cyclisation pattern: clade I (C19-ketoreductases responsible for the reduction of pentangular polyketides), clade II (C17-ketoreductases from the anthracycline biosynthesis), clade III (C15-ketoreductases from the biosynthesis of angucycline antibiotics), and clade IV (specific C9-ketoreductases from the biosynthesis of benzoisochromane quinines such as actinorhodin and granaticin, anthracyclines such as aclacinomycin, and angucyclines such as jadomycin and urdamycin). Clades I–III form a superfamily that reduces a carbonyl group in either terminal or ring positions. Based on sequence similarity (data not shown). MsnO11 belongs to clade IV, whereas MsnO1 and MsnO10 are members of clade II. To investigate the functions of the ketoreductases of the mensacarcin cluster, gene inactivation experiments were performed. Analysis of the metabolic profiles of the resulting strains revealed the production of new compounds. S. albus containing cos2DmsnO1 and pKR1 produced MsnKH1 (3, m/z 367.2 [M H] ; Scheme 1). The structure of MsnKH1 is identical to that of SEK43.[19] SEK43 was described as a shunt product of the polyketide chain generated by the minimal PKS and a C9ketoreductase. Thus MsnO1 seems to act after C9-ketoreduction. Deletion of msnO11 (proposed C9-ketoreductase) led to complete loss of DDMM biosynthesis, and no shunt product
Table 1. Inactivation mutants of S. albus::cos2 and the produced intermediates. Inactivated gene(s) Description of mutant
Produced intermediate
msnH0 msnH1 msnH4 msnH0-H4 msnH6 msnH7 msnC1 msnC2 msnC3 msnO1 msnO2 msnO3
S. albus::cos2DO1/pKR1 S. albus::cos2DH1/pKR1 S. albus::cos2DH4/pKR1 S. albus::cos2DH0-H4/pKR1 S. albus::cos2DH6/pKR1 S. albus::cos2DH7/pKR1 S. albus::cos2DC1/pKR1 S. albus::cos2DC2/pKR1 S. albus::cos2DC3/pKR1 S. albus::cos2DO1/pKR1 S. albus::cos2DO2/pKR1 S. albus::cos2DO3/pKR1
msnO4 msnO5 msnO6 msnO7 msnO8 msnO9 msnO10 msnO11 msnR1
S. albus::cos2DO4/pKR1 S. albus::cos2DO5/pKR1 S. albus::cos2DO6/pKR1 S. albus::cos2DO7/pKR1 S. albus::cos2DO8/pKR1 S. albus::cos2DO9/pKR1 S. albus::cos2DO10/pKR1 S. albus::cos2DO11/pKR1 S. albus::cos2DR1
DDMM (2) DDMM (2) DDMM (2) DDMM (2) –[a] DDMM (2) –[a] –[a] –[a] MsnKH1 (3) DDMM (2), MsnSM3 (7) MsnSM3 (7), MsnSM4 (8), MsnSM5 MsnSM4 (8) MsnKP1 (9), MsnKP4 MsnKP1 (9), MsnKP4 DDMM (2) MsnKP2 (5), MsnKP2a (6) MsnKP1 (9) MsnSM1, MsnSM2 (4) –[a] –[a]
[a] Neither an intermediate nor a shunt product of DDMM was produced.
The ketoreductases MsnO1, MsnO10, and MsnO11 strongly control mensacarcin biosynthesis In PKS biosynthesis ketoreductases catalyze the reduction of a carbonyl group, most often of the C9 carbonyl, to a secondary alcohol. This reaction takes place after the formation of the first ring, typically between C7 C12 or C9 C14. Ketoreductases complement the work of the minimal PKS and promote the correct folding of the polyketide chain. Ketoreductases are also able to affect the orientation of the polyketide chain and thereby the cyclization pattern of the different secondary metabolites derived from PKSII metabolism. One of the best-studied ketoreductases is ActKR (also known as ActIII)[12] from the ChemBioChem 0000, 00, 0 – 0
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was observed. Complementation of the mutant strain with msnO11 recovered production, thus demonstrating that inactivation of msnO11 did not influence the expression of adjacent genes in the cluster. Thus MsnO11, like MsnH6, is essential for mensacarcin biosynthesis. In 2004, Hertweck et al. showed that the minimal PKS activity in enterocin biosynthesis depends on the activity of the ketoreductase EncD.[20] MsnO11 might play a similar role. Deletion of msnO10 led to the production of MsnSM2 (4, m/ z 432.1 [M H] ; Scheme 1). MsnSM2 is a new compound. The polyketide backbone consists of a cyclized heptaketide that carries a methyl group at C12 and a N-acetylcysteine residue at C3. Formation of N-acetylcysteine adducts have been described in the mycothiol-dependent detoxification pathway.[21] Compared to DDMM, MsnMS2 has a shorter chain-length and a different fold of the polyketide chain. The additional methyl group indicates a rearrangement of the polyketide chain during folding. MsnO10 is obviously needed for stabilizing the produced side chain, or even for stabilizing the entire PKS complex.
The luciferase-like monooxygenases MsnO2, MsnO4, and MsnO8 catalyze important tailoring steps in mensacarcin biosynthesis Flavin-dependent monooxygenases form a special group of tailoring enzymes. They catalyze the activation of molecular oxygen by a reduced flavin cofactor, thereby leading to incorporation of oxygen into a molecule.[31] Thus, flavoproteins are able to regio- and/or enantioselectively catalyze different reactions, such as hydroxylations, epoxidations, and Baeyer–Villiger oxidations.[32] Luciferases represent the best-studied group of flavoproteins. They are readily found in bioluminescence organisms such as glowworms, fungi, fish, and bacteria. Luciferins are the substrates for the bioluminescence reaction. Although luciferases catalyze a variety of chemical reactions, the only common factor is the need for oxygen.[33] The luciferase reaction is an oxidative process in which the substrate reaches an electronically excited condition. Upon radiating light, the excited-state intermediate reverts to the ground state.[34] The bacterial luciferase from Vibrio harveyi is a flavin-containing monooxygenase that oxidizes a long-chain aliphatic aldehyde to an aliphatic carboxylic acid while emitting visible light.[34] It forms a heterodimer (a and b subunits) to build a TIM barrel. The single active center is located on the a subunit.[35] The function of luciferase-like monooxygenases in the biosynthesis of streptomycete secondary metabolites has not been described. In the mensacarcin cluster three luciferase-like monooxygenase genes were identified (msnO2, msnO4 and msnO8). We recently showed that inactivation of msnO8 led to the formation of MsnKP2 (5, Scheme 1) and MsnKP2a (6).[4] msnO2 and msnO4 were deleted on cos2 in this study. Deletion of msnO2 led to the production of MsnSM3 (7, m/z 339.2 [M H] ; Scheme 1); deletion of msnO4 led to the production of MsnSM4 (8, m/z 530.1 [M+H] + and 552.1 [M+Na] + ). Surprisingly MsnSM4 retains the carboxyl group at C20, which is not present in MsnSM3. We assume that decarboxylation is somehow inhibited, maybe a consequence of the attached acetylcysteine residue, which is used for detoxification in actinomycetes.[36, 37] Thus, MsnKP2’ (5’; Scheme 2) and MsnSM4’ (8’) are most probably intermediates that become connected to the acetylcysteine residue. Based on the structures of the intermediates, MsnO2 is a dehydratase that introduces the double bond between C12 and C13 of the side
Not even shunt products are produced without cyclases MsnC1, MsnC2, and MsnC3 Ketoreductases, cyclases, and aromatases are needed in addition to the minimal PKS in order to form the backbone of an aromatic polyketide by PKSII. The highly reactive poly-b-intermediate produced by the minimal PKS can spontaneously cyclize. In order to prevent these inadvertent aldol reactions, catalyzing cyclases are needed.[22] Cyclases are cofactor-free enzymes that act in a chaperone-like manner. Although they form a heterogeneous group (in terms of sequence and protein structure), all catalyze specific aldol condensation reactions. Cyclases share the HxGTHxDxPxH motif, which is believed to be part of the active site.[23] Polyketide cyclization is often supported by aromatases, which catalyze the dehydration of cyclic alcohols thus leading to the formation of aromatic ring structures,[24] and by bifunctional cyclases, which can act as aromatases, methyltranferases, or ketoreductases.[23] Three cyclases (MsnC1, MsnC2, and MsnC3) are involved in mensacarcin biosynthesis.[5] Analysis of sequence homology revealed that MsnC1 and MsnC3 share the highest homology with ActVII (actinorhodin pathway) and aromatase ORF4 (granaticin pathway), both responsible for aromatization of the first ring.[12, 25] MsnC2 resembles ORF33 (granaticin biosynthesis)[26] and AlnR (alnumycin pathway), which catalyzes the second ring closure during polyketide biosynthesis.[27] MsnC1, msnC2 and msnC3 were inactivated on cos2, and the mutated cosmids were introduced with pKR1 into S. albus. As expected, no DDMM production was observed. Surprisingly, no shunt product or intermediate was detected. To exclude a systematic error by the inactivation method or effect by the inactivation on the transcription of other genes, each mutant was complemented with the intact cyclase gene; DDMM production was restored in all three cases. There are a number of reports that cyclases are essential for polyketide biosynthesis, but in most cases deletion of the cy-
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Scheme 2. Proposed pathway for mensacarcin biosynthesis showing involvement of mensacarcin enzymes.
chain of mensacarcin. MsnO4 might have multiple roles: introducing two hydroxy groups and one epoxy group into the third ring of mensacarcin. MsnO8 catalyzes epoxidation of the side chain.
sion, MsnO9 seems to be part of the PKS complex and influences the length of the polyketide chain. Three anthron oxygenases, MsnO5, MsnO6, and MsnO7 are involved in mensacarcin biosynthesis
MsnO9 is involved in determining the exact number of acetate units incorporated into the polyketo chain
Anthron oxygenases catalyze the oxidation of naphthacenone and anthron-type precursors of aromatic polyketides to the corresponding quinine derivatives. No cofactor is needed.[43] Anthron oxygenases have been termed “internal oxygenases”, as they use their substrates as reducing equivalents for the reduction of one oxygen to water.[23] A well-known anthron oxygenase is TcmH, which is involved in the conversion of the naphthacenone tetracenomycin F1 to the 5,12-naphthacenquinone tetracenomycin C3 in the biosynthesis of tetracenomycin. Other representatives are ActVA-Orf6 (actinorhodin pathway)[44] and DpgC (vancomycin pathway).[45] The anthron oxygenases in the DDMM pathway exhibit highest homology to AknX (aclacinomycin gene cluster).[46] By in vitro assays with emodineanthron as substrate, AknX was shown to result in the formation of emodineanthraquinone.[47] The genes encoding MsnO5, MsnO6, and MsnO7 were deleted on cos2. Expression of cosmids containing deletions of either msnO5 or msnO6 together with pKR1 resulted in the production of MsnKP1 (9, m/z 285.1 [M H] ; Scheme 1) and MsnKP4. Unfortunately, we were not able to isolate MsnKP4 for NMR studies. Cos2 containing a deleted msnO7 was still able to produce DDMM, however the production rate was considerably lower. As inactivation of msnO5 or msnO6 led to the production of shunt products (MsnKP1 and MsnKP4), this implies independent involvement of both enzymes in the reaction. Both seem to be responsible for the incorporation of oxygen at C5 and each seems to be able to catalyze the reaction. But both en-
BLASTp analysis revealed GrhO7 and ActVI-ORF2 as the closest characterized homologues of MsnO9. These proteins belong to the medium-chain reductases (MDR) superfamily and NADPH:quinone reductases/quinone oxidoreductase subfamily. Reactions catalyzed by NADPH:quinone reductases include the reduction of quinone to a semiquinone radical by transfer of an electron.[38] GrhO7 (griseorhodin biosynthetic gene cluster) was postulated to catalyze the formation of a double bond in dideoxygriseorhodin by dehydrogenation.[39] ActVI-ORF2 (actinorhodin pathway) is known to catalyze the stereospecific 1,4-reduction of (S)-DNPA (4-dihydro-9-hydroxy-1-methyl-10-oxo-3-Hnaphtho-[2,3-c]-pyran-3-(S)-acetic acid) to 5-DDHK (5-deoxy-dihydrokalafungin) at C15.[40] In order to investigate the role of MsnO9, the gene was deleted on cos2. Introduction of deleted cos2 together with pKR1 into S. albus led to the production of MsnKP1 (9, m/z 285.1 [M H] ; Scheme 1). MsnKP1 is identical to 2-hydroxyislandicin, which was first isolated from the plant Ventilago calyculata by Rao et al,[41] and lacks the side chain of mensacarcin (C15 instead of C19). It has been shown that the minimal PKS from the frenolicin pathway synthesizes polyketide chains of different lengths depending on the enzyme used for co-expression. When the aromatase/cyclase TcmN was used as a coexpression partner, nonaketides were produced. When a ketoreductase from the actinorhodin pathway was the co-expression partner, octa- and nonaketides were produced.[42] In concluChemBioChem 0000, 00, 0 – 0
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carcin, inactivation experiments were performed. Our results indicate that a surprisingly high number of enzymes is involved in determining the length and the folding of the elongated polyketide chain. Luciferase-like monooxygenases are involved in the later steps for the formation of epoxy moieties.
MsnO2, MsnO4, and MsnO8 need MsnO3 for activity
Experimental Section
Flavin-dependent oxidoreductases often need the reduced cofactor FMN for the transfer of oxygen. NAD(P)H flavin-dependent oxidoreductases catalyze the reduction of FMN by NAD(P)H. The transfer of reduced FMN to the oxidoreductase can occur by diffusion or directly by protein–protein interaction. An example of the diffusion process is the two-component enzyme system LuxG/LuxAB from Photobacterium leiognathi. LuxAB, the oxidoreductase, receives reduced FMN from the NADH:FMN oxidoreductase LuxG. Because of the instability of reduced FMN, it was postulated that the affinity to LuxAB is quite high and the binding appears very quick to minimize FMN oxidation and formation of H2O2.[48] Protein–protein transfer interactions have been postulated for the systems in Vibrio fischeri and V. harveyi.[49] We recently showed that MsnO8 activity is dependent on the activity of the NADH-dependent flavin reductase MsnO3.[4] In the present study, expression of cos2 containing deleted msnO3 led to the production of a marginal amount of DDMM as well as MsnSM3 (7, Scheme 1), MsnSM4 (8), and numerous of degradation or shunt products. This indicates that MsnO3 acts in a two-component system to provide reduced FMN to MsnO2, MsnO4, and MsnO8.
l-Red/ET-mediated recombination: Inactivation of genes on the cosmid cos2 was carried out by l-Red/ET-mediated recombination as previously described.[50] The targeted gene was replaced with a spectinomycin-resistance cassette, which was subsequently removed by restriction digestion with NdeI followed by ligation. Insertion and removal of the spectinomycin cassette and intergeneric conjugation were as previously described.[4] Insertion of the spectinomycin cassette and inactivation of the gene were verified by PCR (primer sequences for the amplification of the spectinomycin cassettes and verification are in the Supporting Information). Cloning of genes for complementation: For generating the complementation plasmids of msnO1, msnO4–msnO11 and msnR1, pUWL-H-tnp[51, 52] was used as the expression vector. pUWL-H-tnp was linearized by restriction digestion with BamHI and HindIII. All genes were amplified by PCR from cos2,[53] with forward and reverse primers (sequences given in the Supporting Information) containing HindIII and BamHI restriction sites. The PCR products were restriction digested and ligated into the linearized pUWL-Htnp. After successful insertion, the constructed plasmid was digested with BamHI and SpeI. MsnR1 was amplified by PCR with primers F-SARP and R-SARP (incorporating BglII and SpeI restriction sites). The ligation of this PCR product into the previously constructed plasmids resulted in plasmids pKO4R1–pKO11R1. For the construction of pKO2R1, pKRO3R1, pKRC1R1–pKRC3R1 and pKH6R1, the first step was to construct pKR1 from pUWL-H-tnp. For this, msnR1 was amplified from cos2 with primers pKR1-F and pKR1-R and cloned at HindIII and SpeI sites into pUWL-H-tnp. msnO2, msnO3, msnC1-C3, and msnH6 were amplified by PCR from cos2 and digested (restriction sites ClaI and HindIII in the primers). pKR1 was similarly digested and ligated to the PCR products. As msnH7 is directly upstream of msnR1 in the msn cluster, these genes were amplified from cos2 as one PCR product (primers KR1-F and KH7-R). This and pUWL-H-tnp were each cut with HindIII and SpeI, and ligated to obtain pKRH7R1.
Proposed biosynthesis pathway of mensacarcin Mensacarcin (1) is constructed from 10 acetate units. An enzyme complex consisting of the minimal PKS (MsnK1, MsnK2, and MsnK3), the C9-ketoreductase (MsnO11), three cyclases (MsnC1, MsnC2, and MsnC3), and a short-chain dehydrogenase (MsnH6) is required for the formation of a polyketo derivative. In addition, two ketoreductases (MsnO1 and MsnO11) and the medium-chain instead of dehydrogenase/reductase (MsnO9) are involved in determining the exact number of acetate units and the folding of the polyketide chain. The anthron oxygenases (MsnO5 and MsnO6, but not MsnO7) also have an impact on the chain length of the polyketide chain; the thioesterase (MsnH7) catalyzes the release of the polyketide from the ACP. The first correctly folded intermediate containing all carbons is MsnSM3 (7). MsnO2 converts it into MsnSM4’ (8’), which is converted into MsnKP2’ (5’) by MsnO4. Finally, the introduction of the epoxy group of the side chain is catalyzed by MsnO8. MsnO2, msnO4, and MsnO8 require the activity of MsnO3 for full activity (Scheme 2).
Production and analysis of didesmethylmensacarcin and intermediates: The cosmids and plasmids were transferred by intergeneric conjugation into S. albus strain J1074.[54] The exconjugants were cultured for 24 h in tryptic soy broth (TSB; 30 g L 1) at 28 8C with agitation. This pre-culture was used to inoculate DNPM production medium (soytone (7.5 g L 1), baker’s yeast (5 g L 1), 3-(Nmorpholino)propanesulfonic acid (MOPS, 21 g L 1), pH 6.8). After 96–120 h incubation (28 8C, 180 rpm), the cells were removed by centrifugation, then the medium was adjusted to pH 4 and extracted twice with an equal volume of ethyl acetate. The organic extracts were dried in vacuum and dissolved in methanol for analysis on an 1100 series LC/MS system (254 nm; Agilent Technologies) with electrospray ionization (ESI). The LC system was equipped with a XBridge C-18 column (3.5 mm particle size, 3.9 20 mm; Waters) followed by a second XBridge C-18 column (3.5 mm particle size, 4.6 100 mm). The acetonitrile gradient was as previously described for mensacarcin analysis.[5]
Conclusion Mensacarcin is a potent anticancer agent.[1] It is highly decorated with oxygen and even contains two epoxy moieties. In order to get a deeper insight into the biosynthesis of mensa-
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Purification of MsnKP1: To purify MsnKP1, the supernatant of S. albus::cos2DO9/pKR1 (4 L) was extracted with ethyl acetate. Prepara-
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tive TLC followed by preparative HPLC led to 6.6 mg of pure MsnKP1. The dried extract was dissolved in methanol and applied to a silica gel TLC plate (ADAMANT silica gel 60 F254 ; Macherey– Nagel). The mobile phase was acetic acid (0.05 %) in CH2Cl2/methanol (9:1, v/v). For preparative HPLC an Zorbax-C18-system (Agilent Technologies) was equipped with two columns (5 mm particle size, 50 9.4 mm followed by 5 mm particle size, 150 9.4 mm). The eluent was monitored at 254 and 325 nm with a diode array detector (solvent A: acetonitrile with acetic acid (0.5 % v/v), solvent B: acetic acid (0.5 % v/v); gradient: 40 % A (4 min), linear gradient to 95 % A (11 min), 95 % A (4 min), linear gradient to 40 % A (4 min); flow-rate 2 mL min 1).
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Purification of MsnSM3 and MsnSM4: To purify MsnSM3 and MsnSM4, the supernatant of S. albus::cos2DO4/pKR1 (5 L) was extracted with ethyl acetate. Solid-phase extraction followed by preparative TLC, preparative HPLC, and gel filtration led to 5.5 mg of pure MsnSM3 and 1.5 mg of MsnSM4. The crude extract was fractionated in an Oasis HLB20 35 cc extraction cartridge (Waters) with a methanol/water gradient. Separation by TLC (silica gel 60 F254 ; Machery–Nagel) was performed with acetic acid (0.1 %) in CH2Cl2/ methanol (1:9, v/v). Preparative HPLC was carried out with a system of two Xbridge C18-columns (3.5 mm particle size, 20 4.6 mm followed by 3.5 mm particle size, 100 4.6 mm). The eluent was monitored at 254 nm using a diode array detector (40 % A (4 min), linear gradients to 45 % A (4 min), to 95 % A (10 min), 95 % A (2 min), wash with 40 % A (4 min); flow-rate 0.5 mL min 1. Finally, MsnSM4 was purified by gel filtration on a Sephadex LH20 column (GE Healthcare) with methanol. Purification of MsnSM2: The supernatant of S. albus::cos2DO10/ pKR1 (4 L) was extracted with ethyl acetate. Solid-phase extraction followed by preparative TLC and gel filtration led to pure MsnSM2. The crude extract was fractionated on an Oasis HLB20 35 cc extraction cartridge (Waters) with a methanol/water gradient. Separation by TLC (silica gel 60 F254 ; Machery–Nagel) was with acetic acid (0.5 %) in CH2Cl2/methanol (9:1, v/v). Finally, MsnSM2 was purified by gel filtration on a Sephadex LH20 column with methanol, thereby resulting in 1 mg of MsnSM2. Purification of MsnKH1: The supernatant of S. albus::cos2DO11/ pKR1 (5 L) was extracted with ethyl acetate. Solid-phase extraction followed by preparative TLC, preparative HPLC and gel filtration led to pure MsnKH1. The crude extract was fractionated on an Oasis HLB20 35 cc extraction cartridge with a methanol/water gradient. Separation by TLC (silica gel 60 F254 ; Macherey–Nagel) was with acetic acid (0.5 %) in acetonitrile/water (85:10, v/v). Preparative HPLC was carried out with a system of two Xbridge C18-columns (3.5 mm particle size, 20 3.9 mm followed by 3.5 mm particle size, 100 4.6 mm). The eluent was monitored at 254 nm with a diode array detector (30 % A, linear gradients to 70 % A (8 min), then to 95 % A (1 min), 95 % A (3 min), wash with 30 % A (5 min); flow-rate 0.5 mL min 1). Finally, MsnKH1 was purified by gel filtration on a Sephadex LH20 column with methanol, thereby resulting in 5.1 mg of MsnKH1. Structure elucidation: NMR spectra were measured on a VNMR-S 600 spectrometer (1H: 600 MHz, 13C: 150 MHz; Varian) at 35 8C ([D6]DMSO) or 25 8C (other solvents). Residual solvent signals were used as internal references ([D6]DMSO: dH = 2.50 ppm, dC = 39.5 ppm; CD3OD: dH = 3.30 ppm, dC = 49.0 ppm). HR-ESI-MS were recorded on an LTQ Orbitrap XL (Thermo Scientific) and an Apex-Q III (Bruker). MsnSM4 was analyzed on a model 1100 HPLC/ESI-MS device (Agilent Technologies) coupled to a MSD.
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FULL PAPERS MsnO2, MsnO4, and MsnO8: We elucidated the components of the Streptomyces bottropensis mensacarcin biosynthesis gene cluster by selective deletion and complementation in a heterologous expression system. Three luciferase-like monooxygenases are involved in later steps of mensacarcin biosynthesis.
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S. Maier, T. Heitzler, K. Asmus, E. Brçtz, U. Hardter, K. Hesselbach, T. Paululat, A. Bechthold* && – && Functional Characterization of Different ORFs Including LuciferaseLike Monooxygenase Genes from the Mensacarcin Gene Cluster
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