Department of Biochemistry and Food Chemistry, University of Turku, Vatselankatu 2,FIN-20014, Turku, Finland1
Galilaeus Oy, PO Box 113, FIN-20781, Kaarina, Finland2
Author for correspondence: Kaj Räty. Tel: +358 2 333 6856. Fax: +358 2 333 6860. e-mail: kaj.raty{at}utu.fi
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: anthracycline, complementation, deoxyhexose pathway
Abbreviations: Akv, aklavinone; dF, 2-deoxyfucose; NTG, N-methyl-N'-nitro-N-nitrosoguanidine; PKS, polyketide synthase; Rho, rhodinose; Rhn, rhodosamine
a Present address: Lividans Oy, Lemminkäisenkatu 30, FIN-20520 Turku, Finland.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
The biosynthetic pathway of Akv starts by the condensation of one propionate and nine acetates in a reaction series catalysed by the minimal PKS (Fig. 2a) (see reviews by Hutchinson, 1997
; Strohl et al., 1997
). Subsequently, ketoreduction, cyclization and oxygenation reactions in the PKS complex lead to a stable intermediate, aklanonic acid. Post-polyketide reactions, such as methylation of the carboxylic acid, cyclization and reduction, result in Akv, which is ready for glycosylation. Three deoxysugars synthesized in S. galilaeus are attached to position C-7 of Akv in the following sequence: Rhn, dF and rhodinose (Rho) (resulting in AcmN). The attached Rho is rapidly converted by an extracellular oxidoreductase to cinerulose A (to form AcmA) and further to aculose (to form AcmY) (Yoshimoto et al., 1979
). The third sugar residue is further converted to cinerulose B to form AcmB, which is then taken into the bacterial cell and converted back to AcmA (Gräfe et al., 1988
).
We have previously described an S. galilaeus ATCC 31615 mutant series blocked at different steps of the production of aclacinomycin A by N-methyl-N'-nitro-N-nitrosoguanidine (NTG) mutagenization (Ylihonko et al., 1994 ). The mutants were characterized on the basis of their products and biotransformations. Cloning of the genes for aclacinomycins (Räty et al., 2000
) and nogalamycin, another anthracycline (Torkkell et al., 2001
, 1997
; Ylihonko et al., 1996a
, b
), facilitated complementation of the mutants. Knowledge of the nature of the mutations clarifies the steps of the biosynthetic pathway for anthracyclines, provides tools for the analysis of gene functions and makes it possible to use the mutants rationally in combinatorial biosynthesis to create novel molecules.
Deoxysugars are in many cases essential for the activity of antibiotics and, thus, knowledge of their biosynthesis is important for rational design of novel active molecules. Therefore, in this study we focused on S. galilaeus strains with altered glycosylation patterns, aiming to identify the mutations leading to a deficient sugar moiety in aclacinomycins. Four strains included in previously described mutant series (Ylihonko et al., 1994 ), H026, H038, H039 and H054, and three additional mutants, H063, H065 (Räty et al., 2000
) and H075 (DSM 11638) generated recently by NTG mutagenization, were investigated (Table 1
, Fig. 2a
). Complementation experiments with DNA fragments derived from S. galilaeus and other anthracycline producers have already given an idea of the biosynthetic genes blocked in strains H039, H054, H063 and H065 (Räty et al., 2000
; Torkkell et al., 2001
, 1997
). Here, we report on a more thorough characterization of the S. galilaeus mutants to clarify the sequence of biosynthetic reactions of the sugar moiety. Mutated genes were revealed by complementation of the mutants with DNA fragments derived from S. galilaeus and further with PCR-amplified single genes. Moreover, corresponding genes from the mutants were amplified to demonstrate the mutation based on the DNA sequence. Finally, these results together with previous ones were used to confirm the mutations and to elucidate the biosynthetic pathway for aclacinomycin sugars, Rhn, dF and Rho.
|
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
DNA isolation and manipulation in E. coli and Streptomyces strains were carried out by standard procedures (Hopwood et al., 1985 ; Sambrook et al., 1989
). All Streptomyces strains were transformed by standard methods (Hopwood et al., 1985
) with minor modifications (Ylihonko et al., 1996a
). The DNA propagated in E. coli was cloned in pIJE486 and introduced into S. lividans TK24 and further into H039, which is easier to transform than the other S. galilaeus strains. Subsequently, DNA isolated from H039 was introduced into the other S. galilaeus mutant strains in question.
PCR.
For expression constructs, genes aknP, aknQ, aknY and aknX2 from S. galilaeus were amplified separately by PCR, using the primers shown in Table 3. Furthermore, the postulated mutated gene was amplified from the corresponding mutant strain. PCR was carried out with 25 pmol each oligonucleotide primer, 1015 ng of the strains chromosomal DNA, 0·2 mM each dNTP, 2% DMSO and 0·7 U DNA polymerase DyNAzyme EXT (Finnzymes) or Pfu (Promega). The template was initially denatured by heating at 96 °C for 3 min, followed by 30 cycles of amplification, i.e. denaturation at 94 °C for 30 s, annealing at 6064 °C for 1 min and extension at 73 °C for 1 min 15 s (with DyNAzyme EXT) or for 2 min 45 s (with Pfu). The reaction was completed with extension for 8·5 min. When Pfu was used, the 3' A-overhangs were generated by additional extension at 73 °C for 10 min with 0·7 U Dynazyme II DNA polymerase (Finnzymes). The PCR products obtained were cloned in E. coli, using the TOPO TA Cloning Kit (Invitrogen) according to the manufacturers instructions.
|
Expression constructs and complementations.
Overlapping S. galilaeus DNA fragments Sg4 and Sg5 from plasmids pSgc4 and pSgc5 (Räty et al., 2000 ) were combined to get the contiguous DNA fragment Sg9 (Table 4
, Fig. 2b
). First, XhoI/NotI-digested Sg5 was cloned into pSL1190 to give pSgc5SL. Second, the NotIBglII portion of Sg4 from NotI/PstI-digested pSgc4 was cloned into pSgc5SL to give Sg9 in pSgc9SL. Subsequently, the Sg9 fragment from XbaI/HindIII-digested pSgc9SL was cloned into pIJE486 and the plasmid was designated pSgs9. Also, XbaI/HindIII-digested Sg5 from pSgc5SL was cloned in pIJE486 to give expression construct pSgs5. Both pSgs5 and pSgs9 were first introduced into S. lividans TK24 and further into S. galilaeus H039, and subsequently into all other S. galilaeus mutants with altered glycosylation patterns: H026, H038, H054, H063, H065 and H075.
Genes aknP, aknQ, aknY and aknX2 in DNA fragments SgP, SgQ, SgY and SgX2, respectively, were amplified by PCR from wild-type S. galilaeus and cloned from pCR2.1-TOPO vectors in pIJE486 under the control of the ermE promoter using XbaI and HindIII restriction sites inserted within primer sequences. Plasmids pSgsP, pSgsQ, pSgsY and pSgsX2 were then introduced, in addition to TK24 and H039, into S. galilaeus mutants thought to contain mutations in the corresponding genes: pSgsP into H075, pSgsQ into H038, pSgsY into H063 and pSgsX2 into H054 and H065.
Detection of anthracycline metabolites.
The strains were cultivated in 30 ml E1 medium for 45 days to determine the nature and amounts of anthracycline metabolites. A 500 µl sample of E1 culture was adjusted to pH 7·0 by addition of 500 µl 1 M potassium phosphate buffer and cells were subsequently extracted with 250 µl methanol and 250 µl toluene. The toluene layer was concentrated and spotted in 10 µl toluene on a precoated Kieselgel 60 F254 glass plate (Merck) developed with toluene/ethyl acetate/methanol/formic acid (50:50:15:3, by vol.). Whenever necessary, metabolites were also detected by HPLC on a Hewlett Packard 1100 series chromatograph equipped with a LiCHroCART RP-18e column (Purospher, 3 µm, 4·0x55 mm) and a diode array detector. The compounds were separated using 0·1% formic acid and 0·1% formic acid in MeCN with a gradient from 70 to 2% formic acid. The flow rate was 1 ml min-1, and detection was done at 254 and 430 nm. The structures of the anthracyclines were deduced from RF values, retention times and UV/VIS-spectra by comparing with references.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Complementation of the mutations in S. galilaeus strains with a set of genes for aclacinomycins (Sg9)
Fragments Sg4 and Sg5, obtained from S. galilaeus gene libraries, were demonstrated previously to contain genes for the deoxyhexose pathway for aclacinomycins (Räty et al., 2000 ). Here, these two fragments were combined to give pSgs9 in pIJE486, carrying a 14·8 kb contiguous DNA sequence for aclacinomycin biosynthesis. Plasmid pSgs9 was introduced into S. galilaeus mutants. Genes in the Sg9 fragment complemented the mutation in H063 completely and in H054 and H065 partially, as expected, because the mutations were earlier complemented similarly by the introduction of pSgs4 (Räty et al., 2000
). In addition, mutations in H038 and H075 were fully complemented by the introduction of plasmid pSgs9 to restore the wild-type production profile. In contrast, introduction of pSgs9 into mutants H026 and H039 did not have any effect on anthracycline metabolites of the strains.
Mutations in S. galilaeus strains
Mutations in strains H038, H054, H063, H065 and H075, complemented by the introduction of plasmid pSgs9, were studied more closely. The gene that was concluded to be responsible for the complementation of the corresponding mutant, as deduced from the putative gene product and production profile of the mutant, was amplified by PCR from the wild-type S. galilaeus, sequenced and expressed in the mutant. Furthermore, the corresponding gene was amplified by PCR from the mutant strain and its mutation was analysed by comparing its DNA sequence to that of the wild-type S. galilaeus.
H038. The endogenous product of H038 is AcmT, an intermediate of aclacinomycin A biosynthesis, with Rhn as its only sugar. The mutation in H038 was thus concluded to be in a gene which participates in the biosynthesis or transfer of both dF and Rho. In addition to pSgs9, introduction of its subclone pSgs5 into H038 restored wild-type production in the mutant. Thus, based on the proposed hypothetical pathway, aknQ, encoding dTDP-hexose 3-ketoreductase, was the most probable candidate for the mutated gene. Consistently, expression of aknQ in H038 restored wild-type production in the mutant.
AknQ has significant amino acid homology to several dTDP-hexose 3-ketoreductases from antibiotic clusters containing 2-deoxyhexose sugars. Recently, in vitro studies of AknQ homologues Gra Orf26 (identity/similarity 48/61%) from the Streptomyces violaceoruber Tü22 granaticin cluster (Ichinose et al., 1998 ) and Tü99 Orf11 (51/64%) from the Streptomyces antibioticus Tü99 oleandomycin cluster (Draeger et al., 1999
) illustrated their participation in the 2-deoxygenation step (Draeger et al., 1999
). These enzymes were shown to operate in concert with dTDP-4-keto-deoxyglucose 2,3-dehydratases by stabilizing their labile 2-deoxygenated product through 3-ketoreduction.
Sequencing of the mutated aknQ from H038 revealed a G-to-A transition at nucleotide 524, causing a glycine-to-aspartate substitution at amino acid 175. This glycine is conserved in the C-terminal /ß domain of the GFO_IDH_MocA oxidoreductase family (pfam02894) and, furthermore, it is the first in a set of four identical amino acids, GGAL, in AknQ and its closest homologues. This missense mutation prevents the biosynthesis of both Rho and dF in H038 and was complemented by the expression of aknQ.
H063. Mutant strain H063 accumulates Akv. This indicates the mutation to be in a gene catalysing one of the first steps in the biosynthetic pathway of deoxyhexoses or in a glycosyltransferase. Recently, the mutation in H063 was complemented with pSgs44, carrying genes only for cyclase (aknW), aminomethylase (aknX2), dTDP-glucose-1-synthase (aknY) and partial aminotransferase (aknZ) (Räty et al., 2000 ). Since dTDP-glucose-1-synthase catalyses the well-established first step of the deoxyhexose pathway, the mutation was suggested to be in gene aknY. Here, as expected, expression of PCR-amplified aknY in H063 complemented the mutation in the strain.
The DNA sequence of aknY in S. galilaeus H063 demonstrated a C-to-T transition mutation at position 53, resulting in a threonine-to-isoleucine change at amino acid 18 in the conserved nucleotidyl transferase domain (pfam00483). The mutation prevents the biosynthesis of deoxyhexoses completely, because no detectable amount of glycosidic metabolites was recovered from the culture broth of H063, but complementation with aknY was complete.
H075. In addition to its main product Akv-Rhn-dF-dF, H075 produces minor amounts of Akv-dF-dF-dF. Thus, the mutation in H075 would be predicted to be in a gene taking part in the biosynthesis or transfer of Rho. Very recently, dTDP-hexose 3-dehydratase, encoded by rdmI, from rhodomycin-producing Streptomyces purpurascens ATCC 25489, was revealed to complement the mutation in strain H075 (data not shown). Here, plasmid pSgs5, which includes aknP for dTDP-hexose 3-dehydratase, also complemented mutation in H075. Consistently, expression of aknP restored the wild-type production profile in H075.
AknP, a polypeptide of 434 aa, resembles several putative 3-dehydratases involved in the C-3 deoxygenation step in the deoxysugar biosynthesis of antibiotics. The closest homologues are UrdQ (identity/similarity 72/81%) from the Streptomyces fradiae Tü2717 urdamycin biosynthetic gene cluster (Hoffmeister et al., 2000 ), LanQ (71/81%) from the Streptomyces cyanogenus S136 landomycin cluster (Westrich et al., 1999
) and the gra-ORF23 product (71/82%) from the Streptomyces violaceoruber Tü22 granaticin cluster (Ichinose et al., 1998
; Tornus & Floss, 2001
). Moreover, AknP is similar to CDP-4-keto-6-deoxyglucose-3-dehydratases RfbH and AscC (E1) participating in the deoxysugar biosynthesis of the O-antigen in Yersinia pseudotuberculosis (Kessler et al., 1993
; Lei et al., 1995
). AscC and RfbH are pyridoxamine 5'-phosphate (PMP)-dependent iron-sulphur-containing enzymes, which catalyse the CO bond cleavage at C-3 of the substrate, leading to the formation of 3,6-dideoxyhexose. All above-mentioned 3-dehydratases share the Gx3Dx7Ax8EDx10Gx3Gx13Hx4GEGGx19Gx2Cx1Cx7C motif, which is similar to the secondary metabolic aminotransferase family (SMAT) (Piepersberg, 1994
). However, 3-dehydratases differ from their proposed ancestors, pyridoxal 5'-phosphate-dependent aminotransferases, in having a conserved histidine residue at the active site, instead of lysine, indicating dependence of 3-dehydratases on PMP (Lei et al., 1995
). In addition, iron-sulphur-binding cysteine residues are not found in the aminotransferases.
Sequencing of the mutated aknP from H075 revealed a G-to-A transition at codon 245 that changes tryptophan (TGG) to a stop codon (TGA). The mutation precedes the last conserved glycine in the 3-dehydratase motif. As is typical of nonsense mutations, 3-dehydratase enzyme activity is fully missing in H075, resulting in products without Rho.
H054 and H065. Both H054 and H065, whose production profiles are not the same but are very similar, accumulate anthracyclines consisting of only neutral sugars, thus being deficient in the biosynthesis or transfer of the aminosugar Rhn. Instead, a different combination of Rho, dF and cinerulose A, either as di- or trisugar moieties, is produced. In an earlier work, mutations in H054 and H065 were partially complemented with plasmid pSgs44, carrying genes for a cyclase (aknW), an aminomethylase (aknX2), a dTDP-glucose-1-synthase (aknY) and a part of an aminotransferase lacking the C terminus (aknZ) (Räty et al., 2000 ). Of these genes, aknX2 and aknZ participate in the biosynthesis of Rhn solely. The incompleteness of gene aknZ led us to the misassumption that the mutated gene in strains H054 and H065 is the aminomethylase (aknX2). In this study, however, the introduction of plasmid pSgsX2 carrying aknX2 into mutants H054 and H065 did not alter their production profiles. Furthermore, there were no nucleotide changes in the sequence of aknX2 in either mutant when compared to that of the wild-type gene.
According to our results, the complementing gene has to be the one encoding aminotransferase, although it is only partial in plasmid pSgs44. Therefore, the expression construct pSgs45 (Räty et al., 2000 ) (Table 2
, Fig. 2b
), containing partial aknZ with aknY, which encodes dTDP-glucose-1-synthase, was introduced into H054 and H065. Both mutants were partly complemented, as was noted with pSgs44. Thus, the truncated AknZ seems to be capable of partial complementation of the mutations in H054 and H065. The length of the product of partially cloned aknZ is 340 aa, whereas a corresponding gene, aclZ, from another aclacinomycin-producing S. galilaeus (mutant strain 3AR-33, which is derived from strain ATCC 31133) encodes a polypeptide of 369 aa (96/97%; GenBank, accession no. AB008466). The lack of about 30 aa in AknZ leads to weakened, but not abolished, aminotransferase activity, enabling partial complementation of mutations in H054 and H065.
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Strain H063, accumulating Akv, was shown to contain a missense mutation in the putative dTDP-glucose-1-synthase gene aknY. dTDP-glucose-1-synthase adds a deoxynucleotide to a phosphorylated glucose, catalysing the common and well-established first step in the deoxyhexose pathway. Deficiency in this enzyme explains the lack of any deoxysugars in H063 products. In addition, the complementation of H063 with aknY strengthens the previous assignation of AknY function.
In strain H038, a missense mutation in the dTDP-hexose 3-ketoreductase gene aknQ was seen to result in accumulation of AcmT, with Rhn as its only sugar. This strengthens the previous assumption that biosynthetic routes of Rho and dF diverge from that of Rhn in the 2-deoxygenation step (Räty et al., 2000 ). In vitro studies with purified enzymes have clarified the mechanism of the 2-deoxygenation step of neutral deoxyhexoses (Chen et al., 1999
; Draeger et al., 1999
). It was illustrated with enzymes derived from the granaticin and oleandomycin clusters that 2,3-dehydratase in concert with 3-ketoreductase converts dTDP-4-keto-6-deoxy-D-glucose into dTDP-4-keto-2,6-dideoxy-D-glucose (Draeger et al., 1999
). We assume that 3-ketoreduction in the deoxysugar biosynthetic pathway of aclacinomycin results similarly in dTDP-4-keto-2,6-dideoxy-D-glucose with the equatorial (3R) hydroxyl, although 3-ketoreduction in mycarose biosynthesis has been shown to lead to opposite stereochemistry (Chen et al., 1999
). This is because AknQ is more similar to 3-ketoreductases Gra Orf26 and Tü99 Orf11 derived from the granaticin and oleandomycin biosynthetic clusters, respectively. Furthermore, both AknQ and Gra Orf26 are involved in the biosynthesis of Rho.
The mutation in H075, which accumulates aclacinomycins without Rho or its derivatives, was shown to be a nonsense mutation in the gene encoding dTDP-hexose 3-dehydratase, aknP. This is in accordance with the previous assumption that the biosynthesis of Rho differs from that of dF in the 3-deoxygenation step (Räty et al., 2000 ).
Mutants H054 and H065, which accumulate only aclacinomycins with neutral sugars, were shown to be deficient in the aminotransferase AknZ. Also, complementation experiments with genes derived from the nogalamycin biosynthetic cluster support this conclusion (Torkkell, 2001 ). Mutations in H054 and H065 were complemented by snogI, encoding a 370 aa aminotransferase for nogalamine, a sugar moiety of nogalamycin. The absence of approximately 30 aa in AknZ does not abolish all of its catalytic activity, whilst it explains why the mutations were only partially complemented. As discussed above, the biosynthetic pathway of Rhn differs from that of Rho and dF in the 2-deoxygenation step. Instead of 3-ketoreductase, the product of dTDP-4-keto-6-deoxyglucose 2,3-dehydratase is probably stabilized by an aminotransferase (AknZ), as has been suggested for other aminosugars (Draeger et al., 1999
; Olano et al., 1999
).
Apart from the above-mentioned mutants complemented with akn genes, the mutation in strain H039 has been complemented partially with a dTDP-4-keto-6-deoxyhexose reductase gene, snogG, derived from the nogalamycin biosynthetic cluster (Torkkell et al., 2001 , 1997
). Since the sugar moieties of aclacinomycins produced by H039 consist of Rho residues only, inactivation of 4-ketoreductase seems to prevent the biosynthesis of both Rhn and dF. Thus, according to the proposed pathway, the same 4-ketoreductase catalyses the reduction of both dF and Rhn. Very recently, a homologue of snogG, aknM, was also found in the S. galilaeus aclacinomycin cluster (Räty et al., 2002
). However, aknM is only partially in the cloned region and did not complement the mutation in strain H039 (data not shown). Expression of another dTDP-4-keto-6-deoxyhexose reductase gene from the nogalamycin biosynthetic cluster, snogC, producing the inverted (4S) configuration, resulted in the production of Akv-4'-epi-dF in H039 (Torkkell et al., 2001
). SnogC probably acts on the 2-deoxygenated sugar dTDP-4-keto-2,6-dideoxy-D-glucose in aclacinomycin deoxyhexose biosynthesis, preventing the 3-deoxygenation step in the biosynthesis of Rho, but allowing epimerization of the dF route. Epimerization might also precede 3-deoxygenation and would thus be a common step in the biosynthesis of Rho and dF. However, the synthesis of D-Rho in S. fradiae mutant RN-435, which produces urdamycin M, implies that in Rho biosynthesis 3-deoxygenation occurs prior to 5-epimerization (Hoffmeister et al., 2000
).
The mutation in H026 has not yet been complemented. However, the production of Akv-Rhn-dF-Rho strongly implies that H026 is deficient in an oxidoreductase which converts attached Rho into cinerulose A (resulting in AcmA) and further into L-aculose (AcmY) (Yoshimoto et al., 1979 ). This conclusion is supported by the capability of H026 to convert fed AcmB into AcmA, but not further into AcmY (Ylihonko et al., 1994
).
The diversity of aclacinomycin glycosylation in S. galilaeus mutants implies loose substrate specificity of glycosyltransferases. Although Rhn appears only as the first sugar in aclacinomycins produced by the mutants, dF and Rho can be found in different combinations as first, second or third sugar. Furthermore, 4'-epi-dF was attached to Akv when the dTDP-4-keto-6-deoxyhexose reductase gene from the nogalamycin biosynthetic cluster, snogC, was expressed in the H039 mutant (Torkkell et al., 2001 ). Recently, relaxed specificity for both sugar co-substrates and alcohol substrates has been described for various glycosyltransferases (Aguirrezabalaga et al., 2000
; Blanco et al., 2001
; Hoffmeister et al., 2000
; Tang & McDaniel, 2001
; Trefzer et al., 2001
; Zhao et al., 1999
). So far, two glycosyltransferases, AknK and AknS, have been cloned from the S. galilaeus aclacinomycin biosynthetic cluster (Räty et al., 2000
, 2002
). However, their individual function in sugar attachment has not been assigned, and since the whole biosynthetic cluster for aclacinomycins has not yet been cloned, we do not know whether they are involved in the transfer of one or more deoxysugars.
In the present work, we studied more closely the mutations behind the deficient glycosylation of aclacinomycins in S. galilaeus mutants. In each characterized mutant, a single point mutation in a deoxyhexose biosynthetic gene was responsible for abolished enzyme activity leading to the accumulation of products missing one or more of the inherently synthesized deoxysugars. Exact knowledge of the nature of the mutations enhances the usage and value of these mutants in the analysis of gene function and creation of novel compounds. In addition, the results strengthen the assignments of the akn gene products and enlighten the biosynthetic pathway for deoxyhexoses. These results also show that chemical mutagenization, despite its randomness, still offers a convenient tool to create single point mutations affecting only a specific biosynthetic gene, and that it can be used together with molecular biology to study the biosynthesis of different compounds and to learn more about features important to the catalytic activity of the enzymes.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bibb, M. J., Janssen, G. R. & Ward, J. M. (1985). Cloning and analysis of the promoter region of the erythromycin resistance gene (ermE) of Streptomyces erythraeus. Gene 38, 215-226.[Medline]
Blanco, G., Patallo, E. P., Braña, A. F., Trefzer, A., Bechthold, A., Rohr, J., Méndez, C. & Salas, J. A. (2001). Identification of a sugar flexible glycosyltransferase from Streptomyces olivaceus, the producer of the antitumor polyketide elloramycin. Chem Biol 8, 253-263.[Medline]
Casey, M. L., Paulick, R. C. & Whitlock, H. W. (1978). Carbon-13 nuclear magnetic resonance study of the biosynthesis of daunomycin and islandicin. J Org Chem 43, 1627-1634.
Chen, H., Agnihotri, G., Guo, Z., Que, N. L. S., Chen, X. H. & Liu, H.-w. (1999). Biosynthesis of mycarose: isolation and characterization of enzymes involved in the C-2 deoxygenation. J Am Chem Soc 121, 8124-8125.
Draeger, G., Park, S.-H. & Floss, H. G. (1999). Mechanism of the 2-deoxygenation step in the biosynthesis of the deoxyhexose moieties of the antibiotics granaticin and oleandomycin. J Am Chem Soc 121, 2611-2612.
Eckardt, K., Schumann, G., Gräfe, U., Ihn, W., Wagner, C., Fleck, W. F. & Thrum, H. (1985). Preparation of labeled aklanonic acid and its bioconversion to anthracyclinones by mutants of Streptomyces griseus. J Antibiot 38, 1096-1097.[Medline]
Fujii, I. & Ebizuka, Y. (1997). Anthracycline biosynthesis in Streptomyces galilaeus. Chem Rev 97, 2511-2523.[Medline]
Gräfe, U., Dornberger, K., Fleck, W. F. & Freysoldt, C. (1988). Compartmentation of enzymes interconverting aclacinomycins in Streptomyces species AM 33352. J Basic Microbiol 28, 17-23.[Medline]
Grein, A. (1987). Antitumor anthracyclines produced by Streptomyces peucetius. Adv Appl Microbiol 32, 203-214.[Medline]
Hoffmeister, D., Ichinose, K., Domann, S. & 9 other authors (2000). The NDP-sugar co-substrate concentration and the enzyme expression level influence the substrate specificity of glycosyltransferases: cloning and characterization of deoxysugar biosynthetic genes of the urdamycin biosynthetic gene cluster. Chem Biol 7, 821831.[Medline]
Hopwood, D. A., Bibb, M. J., Chater, K. F. & 7 other authors (1985). Genetic Manipulation of Streptomyces: a Laboratory Manual. Norwich: John Innes Foundation.
Hutchinson, C. R. (1997). Biosynthetic studies of daunorubicin and tetracenomycin C. Chem Rev 97, 2525-2535.[Medline]
Ichinose, K., Bedford, D. J., Tornus, D., Bechthold, A., Bibb, M. J., Revill, W. P., Floss, H. G. & Hopwood, D. A. (1998). The granaticin biosynthetic gene cluster of Streptomyces violaceoruber Tü22: sequence analysis and expression in a heterologous host. Chem Biol 5, 647-659.[Medline]
Kessler, A. C., Haase, A. & Reeves, P. R. (1993). Molecular analysis of the 3,6-dideoxyhexose pathway genes of Yersinia pseudotuberculosis serogroup IIA. J Bacteriol 175, 1412-1422.[Abstract]
Kieser, T., Bibb, M. J., Buttner, M. J., Chater, K. F. & Hopwood, D. A. (2000). Practical Streptomyces Genetics. Norwich: John Innes Foundation.
Kitamura, I., Tobe, H., Yoshimoto, A., Oki, T., Naganawa, H., Takeuchi, T. & Umezawa, H. (1981). Biosynthesis of aklavinone and aclacinomycins. J Antibiot 34, 1498-1500.[Medline]
Lei, Y., Ploux, O. & Liu, H.-w. (1995). Mechanistic studies on CDP-6-deoxy-L-threo-D-glycero-4-hexulose 3-dehydrase: identification of His-220 as the active-site base by chemical modification and site-directed mutagenesis. Biochemistry 34, 4643-4654.[Medline]
Oki, T., Matsuzawa, Y., Yoshimoto, A. & 10 other authors (1975). New antitumor antibiotics, aclacinomycins A and B. J Antibiot 28, 830834.[Medline]
Olano, C., Lomovskaya, N., Fonstein, L., Roll, J. T. & Hutchinson, C. R. (1999). A two-plasmid system for the glycosylation of polyketide antibiotics: bioconversion of -rhodomycinone to rhodomycin D. Chem Biol 6, 845-855.[Medline]
Paulick, R. C., Casey, M. L. & Whitlock, H. W. (1976). A 13C nuclear magnetic resonance study of the biosynthesis of daunomycin from 13CH313CO2Na. J Am Chem Soc 98, 3370-3371.[Medline]
Piepersberg, W. (1994). Pathway engineering in secondary metabolite-producing actinomycetes. Crit Rev Biotechnol 14, 251-285.[Medline]
Räty, K., Kunnari, T., Hakala, J., Mäntsälä, P. & Ylihonko, K. (2000). A gene cluster from Streptomyces galilaeus involved in glycosylation of aclarubicin. Mol Gen Genet 264, 164-172.[Medline]
Räty, K., Kantola, J., Hautala, A., Hakala, J., Ylihonko, K. & Mäntsälä, P. (2002). Cloning and characterization of Streptomyces galilaeus aclacinomycins polyketide synthase (PKS) cluster. Gene 293, 115-122.[Medline]
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Schumann, G., Stengel, C., Eckardt, K. & Ihn, W. (1986). Biotransformation of aklanonic acid by blocked mutants of anthracycline-producing strains of Streptomyces galilaeus and Streptomyces peucetius. J Basic Microbiol 26, 249-255.[Medline]
Strohl, W. R., Dickens, M. L., Rajgarhia, V. B., Woo, A. J. & Priestley, N. D. (1997). Anthracyclines. In Biotechnology of Antibiotics , pp. 577-657. Edited by W. R. Strohl. New York:Marcel Dekker Inc.
Tang, L. & McDaniel, R. (2001). Construction of desosamine containing polyketide libraries using a glycosyltransferase with broad substrate specificity. Chem Biol 8, 547-555.[Medline]
Torkkell, S. (2001). Anthracycline antibiotics: biosynthetic pathway and molecular genetics of nogalamycin, a product of Streptomyces nogalater. PhD thesis, University of Turku, Finland.
Torkkell, S., Ylihonko, K., Hakala, J., Skurnik, M. & Mäntsälä, P. (1997). Characterization of Streptomyces nogalater genes encoding enzymes involved in glycosylation steps in nogalamycin biosynthesis. Mol Gen Genet 256, 203-209.[Medline]
Torkkell, S., Kunnari, T., Palmu, K., Mäntsälä, P., Hakala, J. & Ylihonko, K. (2001). The entire nogalamycin biosynthetic gene cluster of Streptomyces nogalater: characterization of a 20-kb DNA region and generation of hybrid structures. Mol Genet Genomics 266, 276-288.[Medline]
Tornus, D. & Floss, H. G. (2001). Identification of four genes from the granaticin biosynthetic gene cluster of Streptomyces violaceoruber Tü22 involved in the biosynthesis of L-rhodinose. J Antibiot 54, 91-101.[Medline]
Trefzer, A., Fischer, C., Stockert, S., Westrich, L., Künzel, E., Girreser, U., Rohr, J. & Bechthold, A. (2001). Elucidation of the function of two glycosyltransferase genes (lanGT1 and lanGT4) involved in landomycin biosynthesis and generation of new oligosaccharide antibiotics. Chem Biol 8, 1239-1252.[Medline]
Wagner, C., Eckardt, K., Schumann, G., Ihn, W. & Tresselt, D. (1984). Microbial transformation of aklanonic acid, a potential early intermediate in the biosynthesis of anthracyclines. J Antibiot 37, 691-692.[Medline]
Ward, J. M., Janssen, G. R., Kieser, T., Bibb, M. J., Buttner, M. J. & Bibb, M. J. (1986). Construction and characterization of a series of multicopy promoter-probe plasmid vectors for Streptomyces using the aminoglycoside phosphotransferase from Tn5 as indicator. Mol Gen Genet 203, 468-478.[Medline]
Westrich, L., Domann, S., Faust, B., Bedford, D., Hopwood, D. A. & Bechthold, A. (1999). Cloning and characterization of a gene cluster from Streptomyces cyanogenus S136 probably involved in landomycin biosynthesis. FEMS Microbiol Lett 170, 381-387.[Medline]
Ylihonko, K., Hakala, J., Niemi, J., Lundell, J. & Mäntsälä, P. (1994). Isolation and characterization of aclacinomycin A-non-producing Streptomyces galilaeus (ATCC 31615) mutants. Microbiology 140, 1359-1365.[Abstract]
Ylihonko, K., Hakala, J., Kunnari, T. & Mäntsälä, P. (1996a). Production of hybrid anthracycline antibiotics by heterologous expression of Streptomyces nogalater nogalamycin biosynthesis genes. Microbiology 142, 1965-1972.[Abstract]
Ylihonko, K., Tuikkanen, J., Jussila, S., Cong, L. & Mäntsälä, P. (1996b). A gene cluster involved in nogalamycin biosynthesis from Streptomyces nogalater: sequence analysis and complementation of early-block mutations in the anthracycline pathway. Mol Gen Genet 251, 113-120.[Medline]
Ylihonko, K., Hakala, J. & Kunnari, T. (1999). Hybrid anthracyclines from genetically engineered Streptomyces galilaeus strains. International Patent Application WO 99/58544 A1.
Yoshimoto, A., Ogasawara, T., Kitamura, I., Oki, T., Inui, T., Takeuchi, T. & Umezawa, H. (1979). Enzymatic conversion of aclacinomycin A to Y by a specific oxidoreductase in Streptomyces. J Antibiot 32, 472-481.[Medline]
Zhao, L., Ahlert, J., Xue, Y., Thorson, J. S., Sherman, D. H. & Liu, H.-w. (1999). Engineering a methymycin/pikromycin-calicheamicin hybrid: construction of two new macrolides carrying a designed sugar moiety. J Am Chem Soc 121, 9881-9882.
Received 26 March 2002;
revised 20 June 2002;
accepted 9 July 2002.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
J MED MICROBIOL | ALL SGM JOURNALS |