Biotransformations of anthracyclinones inStreptomyces coeruleorubidus andStreptomyces galilaeus

The ability to transorm biologically exogenous daunomycinone, 13-dihydrodaunomycinone, aklavinone, 7-deoxyaklavinone, epsilon-rhodomycinone, epsilon-isorhodomycinone and epsilon-pyrromycinone was studied in submerged cultures of the following strains: wild Streptomyces coeruleorubidus JA 10092 (W1) and its improved variants 39-146 and 84-17 (type P1) producing glycosides of daunomycinone and of 13-dihydrodaunomycinone, together with epsilon-rhodomycinone, 13-dihydrodaunomycinone and 7-deoxy-13-dihydrodaunomycinone; in five mutant types of S. coeruleorubidus (A, B, C, D, E) blocked in the biosynthesis of glycosides and differing in the production of free anthracyclinones; in the wild Streptomyces galilaeus JA 3043 (W2) and its improved variant G-167 (P2) producing glycosides of epsilon-pyrromycinone and of aklavinone together with 7-deoxy and bisanhydro derivatives of both aglycones; in two mutant types S. galilaeus (F and G) blocked in biosynthesis of glycosides and differing in the occurrence of anthracyclinones. The following bioconversions were observed: daunomycinone leads to 13-dihydrodaunomycinone and 7-deoxy-13-dihydrodaunomycinone (all strains); 13-dihydrodaunomycinone leads to 7-deoxy-13-dihydrodaunomycinone (all strains); daunomycinone or 13-dihydrodaunomycinone leads to glycosides of daunomycinone and of 13-dihydrodaunomycinone, identical with metabolites W1 and P1 (type A), or only a single glycoside of daunomycinone (type E); aklavinone leads to epsilon-rhodomycinone (types A and B); aklaviinone leads to 7-deoxyaklavinone and bisanhydroaklavinone (type C); epsilon-rhodomycinone leads to zeta-rhodomycinone (types C, E); epsilon-rhodomycinone leads to glycosides of epsilon-rhodomycinone (types W2, P2); epsilon-isorhodomycinone leads to glycosides of epsilon-isorhodomycinone (types W2, P2); epsilon-pyrromycinone leads to a glycoside of epsilon-pyrromycinone (types W1, P1). 7-Deoxyaklavinone remained intact in all tests. Exogenous daunomycinone suppressed the biosynthesis of its own glycosides in W1 and P1; it simultaneously increased the production of epsilon-rhodomycinone in P1.     

A gene cluster from Streptomyces galilaeus involved in glycosylation of aclarubicin

We have cloned and characterized a gene cluster for anthracycline biosynthesis from Streptomyces galilaeus. This cluster, 15-kb long, includes eight genes involved in the deoxyhexose biosynthesis pathway, a gene for a glycosyltransferase and one for an activator, as well as two genes involved in aglycone biosynthesis. Gene disruption targeted to the activator gene blocked production of aclacinomycins in S. galilaeus. Plasmid pSgs4, containing genes for a glycosyltransferase (aknS), an aminomethylase (aknX), a glucose-1-phosphate thymidylyltransferase (akn Y) and two genes for unidentified glycosylation functions (aknT and aknV), restored the production of aclacinomycins in the S. galilaeus mutants H063, which accumulates aklavinone, and H054, which produces aklavinone with rhodinose and deoxyfucose residues. Furthermore, pSgs4 directed the production of L-rhamnosyl-epsilon-rhodomycinone and L-daunosaminyl-epsilon-rhodomycinone in S. peucetius strains that produce epsilon-rhodomycinone endogenously. Subcloning of the gene cluster was carried out in order to further define the genes that are responsible for complementation and hybrid anthracycline generation.     

Biosynthesis of anthraquinones by interspecies cloning of actinorhodin biosynthesis genes in streptomycetes: clarification of actinorhodin gene functions.

Streptomyces galilaeus ATCC 31133 and ATCC 31671, producers of the anthracyclines aclacinomycin A and 2-hydroxyaklavinone, respectively, formed an anthraquinone, aloesaponarin II, when they were transformed with DNA from Streptomyces coelicolor containing four genetic loci, actI, actIII, actIV, and actVII, encoding early reactions in the actinorhodin biosynthesis pathway. Subcloning experiments indicated that a 2.8-kilobase-pair XhoI fragment containing only the actI and actVII loci was necessary for aloesaponarin II biosynthesis by S. galilaeus ATCC 31133. Aloesaponarin II was synthesized via the condensation of 8 acetyl coenzyme A equivalents, followed by a decarboxylation reaction as demonstrated by [1,2-13C2]acetate feeding experiments. S. coelicolor B22 and B159, actVI blocked mutants, also formed aloesaponarin II as an apparent shunt product. Mutants of S. coelicolor blocked in several other steps in actinorhodin biosynthesis did not synthesize aloesaponarin II or other detectable anthraquinones. When S. galilaeus ATCC 31671 was transformed with the DNA carrying the actI, actIII, and actVII loci, the recombinant strain produced both aloesaponarin II and aklavinone, suggesting that the actinorhodin biosynthesis DNA encoded a function able to deoxygenate 2-hydroxyaklavinone to aklavinone. When S. galilaeus ATCC 31671 was transformed with a plasmid carrying only the intact actIII gene (pANT45), aklavinone was formed exclusively. These experiments indicate a function for the actIII gene, which is the reduction of the keto group at C-9 from the carboxy terminus of the assembled polyketide to the corresponding secondary alcohol. In the presence of the actIII gene, anthraquinones or anthracyclines formed as a result of dehydration and aromatization lack an oxygen function on the carbon on which the keto reductase operated. When S. galilaeus ATCC 31671 was transformed with the DNA carrying the actI, actVII, and actIV loci, the recombinant strain produced two novel anthraquinones, desoxyerythrolaccin, the 3-hydroxy analog of aloesaponarin II, and 1-O-methyldesoxyerythrolaccin. The results obtained in these experiments together with earlier data suggest a pathway for the biosynthesis of actinorhodin and related compounds by S. coelicolor.     

Cloning, sequencing, and analysis of aklaviketone reductase from Streptomyces sp. strain C5.

DNA sequence analysis of a region of the Streptomyces sp. strain C5 daunomycin biosynthesis gene cluster, located just upstream of the daunomycin polyketide biosynthesis genes, revealed the presence of six complete genes. The two genes reading right to left include genes encoding the potentially translationally coupled gene products, an acyl carrier protein and a ketoreductase, and the four genes reading divergently, left to right, include two open reading frames of unknown function followed by a gene encoding an apparent glycosyltransferase and dauE, encoding aklaviketone reductase. Extracts of Streptomyces lividans TK24 containing recombinant DauE catalyzed the NADPH-specific conversion of aklaviketone, maggiemycin, and 7-oxodaunomycinone to aklavinone, epsilon-rhodomycinone, and daunomycinone, respectively. Neither the product of dauB nor that of the ketoreductase gene directly downstream of the acyl carrier protein gene demonstrated aklaviketone reductase activity.     

Nucleotide sequence of the aknA region of the aklavinone biosynthetic gene cluster of Streptomyces galilaeus.

A 3.4-kb BamHI fragment that is assumed to be a part of the aklavinone biosynthetic gene cluster of Streptomyces galilaeus 3AR-33 and contains the genes required for the early stage of polyketide biosynthesis was sequenced. The nucleotide sequence of the region that hybridizes to the actIII probe reveals the presence of a gene, aknA, whose deduced protein product is very similar to the ActIII protein and other known oxidoreductases. The predicted AknA protein is believed to be responsible for catalyzing the reduction of the keto group at the ninth carbon from the carboxyl terminus of the assembled polyketide to the corresponding secondary alcohol. The predicted AknA protein has a calculated molecular mass of 27,197 Da (261 amino acids) and the highly conserved sequence Gly-Xaa-Gly-Xaa-Xaa-Ala commonly seen in oxidoreductases. Cloning and sequence analysis of the aknA region of the 2-hydroxyaklavinone-producing strain S. galilaeus ANR-58 identified an alteration in the gene, confirming that the aknA gene is essential for aklavinone biosynthesis.     

Survival of Escherichia coli O157:H7 in private drinking water wells: influences of protozoan grazing and elevated copper concentrations

The rdm genes B, C and E from Streptomyces purpurascens encode enzymes that tailor aklavinone and aclacinomycins. We report that in addition to hydroxylation of aklavinone to epsilon-rhodomycinone, RdmE (aklavinone-11-hydroxylase) hydroxylated 11-deoxy-beta-rhodomycinone to beta-rhodomycinone both in vivo and in vitro. 15-Demethoxyaklavinone and decarbomethoxyaklavinone did not serve as substrates. RdmC (aclacinomycin methyl esterase) converted aclacinomycin T (AcmT) to 15-demethoxyaclacinomycin T, which was in turn converted to 10-decarbomethoxyaclacinomycin T and then to rhodomycin B by RdmB (aclacinomycin-10-hydroxylase). RdmC and RdmB were most active on AcmT, the one-sugar derivative, with their activity decreasing by 70-90% on two- and three-sugar aclacinomycins. Aclacinomycin A competitively inhibited the AcmT modifications at C-10. The results presented here suggest that in vivo the modifications at C-10 take place principally after addition of the first sugar.     

Leukaemomycin-blocked mutants of Streptomyces griseus and their pigments

In a continued search for leukaemomycin-blocked mutants of three leukaemomycin-producing strains IMET JA 3933, IMET JA 5142 and IMET JA 5570 of Streptomyces griseus, 32 mutants producing aerial mycelium and spores were detected. Furthermore, in all mutants cosynthetic capability has been observed. This report describes characterization of leukaemomycin-blocked mutants obtained by mutagenic treatment experiments using NTG and combined UV-/X-rays. According to the biosynthetic capability for anthracyclinones or other pigments the mutants could be divided into six classes. The first class contains 14 leukaemomycin-blocked mutants unable to synthesize anthracyclinones. Besides two classes of mutants (12)synthesizing well-known anthracyclinones as epsilon-rhodomycinone, 7-deoxy-epsilon-rhodomycinone, 11-deoxy-derivatives of daunomycinone, three new classes of mutants (6) synthesizing reddish-brown, brown and blue-violet pigments on solid media with structures not elucidated as yet, will be described.     

Genetics of actinorhodin biosynthesis by Streptomyces coelicolor A3(2)

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

Microbial transformation of semisynthetic derivatives of daunomycinone modified in ring a

Semisynthetic derivatives of daunomycinone with 7,9-isopropylacetal, 7-O-methyl, 7-O-(4-penten-2-yl), and 7-O-(2-hydroxyethyl) substituents were converted byStreptomyces peucetius var.caesius (an adriamycin-blocked mutant) into 7-deoxy-13-dihydrodaunomycinone, while daunomycinone was transformed into 13-dihydrodaunomycinone (predominantly) and 7-deoxy-13-dihydrodaunomycinone.S. coeruleorubidus mutants 24–74 (accumulating aclavinone derivatives instead of daunomycin and related compounds) and 96-85 (producing no anthracycline substances), andS. aureofaciens B-96 (a tetracycline-blocked mutant) transformed the above substrates into the corresponding 13-dihydro derivatives, with the exception of 7,9-isopropylacetal daunomycinone which remained intact. 7-O-Propyn-1-yl daunomycinone was not transformed by any of the strains used under the conditions.     

Cloning and characterization of Streptomyces galilaeus aclacinomycins polyketide synthase (PKS) cluster

We have cloned and sequenced polyketide synthase (PKS) genes from the aclacinomycin producer Streptomyces galilaeus ATCC 31,615. The sequenced 13.5-kb region contained 13 complete genes. Their organization as well as their protein sequences showed high similarity to those of other type II PKS genes. The continuous region included the genes for the minimal PKS, consisting of ketosynthase I (aknB), ketosynthase II (aknC), and acyl carrier protein (aknD). These were followed by the daunomycin dpsC and dpsD homologues (aknE2 and F, respectively), which are rare in type II PKS clusters. They are associated with the unusual starter unit, propionate, used in the biosynthesis of aklavinone, a common precursor of aclacinomycin and daunomycin. Accordingly, when aclacinomycins minimal PKS genes were substituted for those of nogalamycin in the plasmid carrying genes for auramycinone biosynthesis, aklavinone was produced in the heterologous hosts. In addition to the minimal PKS, the cloned region included the PKS genes for polyketide ketoreductase (aknA), aromatase (aknE1) and oxygenase (aknX), as well as genes putatively encoding an aklanonic acid methyl transferase (aknG) and an aklanonic acid methyl ester cyclase (aknH) for post-polyketide steps were found. Moreover, the region carried genes for an activator (aknI), a glycosyl transferase (aknK) and an epimerase (aknL) taking part in deoxysugar biosynthesis.     

Mutants ofStreptomyces coeruleorubidus impaired in the biosynthesis of daunomycinone glycosides and related metabolites

Mutants ofStreptomyces coeruleorubidus, blocked in the biosynthesis of anthracycline antibiotics of the daunomycine complex, were isolated from the production strains after treatment with UV light, γ-radiation, nitrous acid, and after natural selection; according to their different biosynthetic activity the mutants were divided into five phenotypic groups. Mutants of two of these groups produced compounds that had not yet been described inStreptomyces coeruleorubidus (aklavinone, 7-deoxyaklavinone, ζ-rhodomycinone and glycosides of ε-rhodomycinone). The mutants differed from the parent strains and also mutually in morphological characteristics but no direct correlation between these changes and the biosynthetic activity could be observed in most cases.     

Cloning and expression of daunorubicin biosynthesis genes from Streptomyces peucetius and S. peucetius subsp. caesius.

Genes for the biosynthesis of daunorubicin (daunomycin) and doxorubicin (adriamycin), important antitumor drugs, were cloned from Streptomyces peucetius (the daunorubicin producer) and S. peucetius subsp. caesius (the doxorubicin producer) by use of the actI/tcmIa and actIII polyketide synthase gene probes. Restriction mapping and Southern analysis of the DNA cloned in a cosmid vector established that the DNA represented three nonoverlapping regions of the S. peucetius subsp. caesius genome. These three regions plus an additional one that hybridized to the same probes are present in the S. peucetius genome, as reported previously (K. J. Stutzman-Engwall and C. R. Hutchinson, Proc. Natl. Acad. Sci. USA 86:3135-3139, 1989). Functional analysis of representative clones from some of these regions in S. lividans, S. peucetius ATCC 29050, S. peucetius subsp. caesius ATCC 27952, and two of its blocked mutants (strains H6101 and H6125) showed that many of the antibiotic production genes reside in the region of DNA represented by the group IV clones. This conclusion is based on the production of epsilon-rhodomycinone, a key intermediate of the daunorubicin pathway, in certain S. lividans transformants and on the apparent complementation of mutations that block daunorubicin biosynthesis in strains H6101 and H6125. Some of the transformants of strains 29050, 27952, and H6125 exhibited substantial overproduction of epsilon-rhodomycinone and daunorubicin.     

Growth and production of anthracyclines in wild-type and mutant strains ofStreptomyces galilaeus

The course of growth curves with respect to the biosynthesis of anthracyclines was followed in the wild low-producing strain Streptomyces galilaeus JA 3043 and in its mutants G-167 (producing increased quantities of glycosides of epsilon-pyrromycinone) and J-14 (accumulating free epsilon-pyrromycinone). A two-phase type of fermentation (growth phase, production phase) was observed in strains JA 3043 and J-14. The maximum production of anthracyclines occurred only after the end of intense growth of the culture. Two phases of rapid growth separated by a phase of stagnation were observed in strain G-167. The second growth phase proceeded only during late hours of cultivation and was (as compared with the first phase) associated with an intensive biosynthesis of anthracyclines.     

Enhanced antibiotic production by manipulation of the Streptomyces peucetius dnrH and dnmT genes involved in doxorubicin (adriamycin) biosynthesis.

Sequence analysis of a 3.4-kb region Streptomyces peucetius daunorubicin (DNR) gene cluster established the presence of the dnrH and dnmT genes. In dnrH mutants, DNR production increased 8.5-fold, compared with that in the wild-type strain, while dnmT mutants accumulated epsilon-rhodomycinone (RHO), which normally becomes glycosylated in daunorubicin biosynthesis. Hence, dnmT may be involved in the biosynthesis or attachment of daunosamine to RHO or in the regulation of this process. Since the DnrH protein is similar to known glycosyl transferases, this protein may catalyze the conversion of DNR to its polyglycosylated forms, known as baumycins. Overexpression of dnmT in the wild-type and dnrH mutant strains resulted in a major decrease in RHO accumulation and increase in DNR production.     

Genome mining in Streptomyces. Elucidation of the role of Baeyer-Villiger monooxygenases and non-heme iron-dependent dehydrogenase/oxygenases in the final steps of the biosynthesis of pentalenolactone and neopentalenolactone

The pentalenolactone biosynthetic gene clusters have been cloned and sequenced from two known producers of the sesquiterpenoid antibiotic pentalenolactone, Streptomyces exfoliatus UC5319 and Streptomyces arenae TU?469. The recombinant enzymes PenE and PntE, from S. exfoliatus and S. arenae, respectively, catalyze the flavin-dependent Baeyer-Villiger oxidation of 1-deoxy-11-oxopentalenic acid (7) to pentalenolactone D (8). Recombinant PenD, PntD, and PtlD, the latter from Streptomyces avermitilis, each catalyze the Fe(2+)-α-ketoglutarate-dependent oxidation of pentalenolactone D (8) to pentalenolactone E (15) and pentalenolactone F (16). Incubation of PenD, PntD, or PtlD with the isomeric neopentalenolactone D (9) gave PL308 (12) and a compound tentatively identified as neopentalenolactone E (14). These results are corroborated by analysis of the ΔpenD and ΔpntD mutants of S. exfoliatus and S. arenae, respectively, both of which accumulate pentalenolactone D but are blocked in production of pentalenolactone as well as the precursors pentalenolactones E and F. Finally, complementation of the previously described S. avermitilis ΔptlE ΔptlD deletion mutant with either penE or pntE gave pentalenolactone D (8), while complemention of the ΔptlE ΔptlD double mutant with pntE plus pntD or penE plus pntD gave pentalenolactone F (16).     

Isolation and sequence analysis of polyketide synthase genes from the daunomycin-producing Streptomyces sp. strain C5.

A contiguous region of about 30 kbp of DNA putatively encoding reactions in daunomycin biosynthesis was isolated from Streptomyces sp. strain C5 DNA. The DNA sequence of an 8.1-kbp EcoRI fragment, which hybridized with actI polyketide synthase (PKS) and actIII polyketide reductase (PKR) gene probes, was determined, revealing seven complete open reading frames (ORFs), two in one cluster and five in a divergently transcribed cluster. The former two genes are likely to encode PKR and a bifunctional cyclase/dehydrase. The five latter genes encode: (i) a homolog of TcmH, an oxygenase of the tetracenomycin biosynthesis pathway; (ii) a PKS Orf1 homolog; (iii) a PKS Orf2 homolog (chain length factor); (iv) a product having moderate sequence identity with Escherichia coli beta-ketoacyl acyl carrier protein synthase III but lacking the conserved active site; and (v) a protein highly similar to several acyltransferases. The DNA within the 8.1-kbp EcoRI fragment restored daunomycin production to two dauA non-daunomycin-producing mutants of Streptomyces sp. strain C5 and restored wild-type antibiotic production to Streptomyces coelicolor B40 (act VII; nonfunctional cyclase/dehydrase), and to S. coelicolor B41 (actIII) and Streptomyces galilaeus ATCC 31671, strains defective in PKR activity.     

Bacterial rep- mutations that block development of small DNA bacteriophages late in infection.

Several related mutants of Escherichia coli C have been isolated that block the growth of the small icosahedral DNA phages phiX174 and S13 late in infection. Phage G6 is also blocked, at a stage not yet known. Growth of the filamentous phage M13, though not blocked, is affected in these strains. These host mutations co-transduce with ilv at high frequency, as do rep- mutations. However, the new mutants, designated groL-, differ from previously studied rep- mutants in that they permit synthesis of progeny replicative-form DNA. The groL- mutants are blocked in synthesis of stable single-stranded DNA of phiX174 and related phages. They are gro+ for P2. Evidence that groL- mutations and rep- mutations are in the same gene is presented. Spontaneous mutants (ogr) of phiX174, S13, and the G phages can grow on groL- strains. The ogr mutations are located in the phage's major capsid gene, F, as determined by complementation tests. There are numerous sites for mutation to ogr. Some mutations in genes A and F interfere with the ogr property when combined with an ogr mutation on the same genome. The ogr mutations are cis acting in a groL- cell; i.e., an ogr mutant gives very poor rescue of a non-ogr mutant. The wild-type form of each G phage appears to be naturally in the ogr mutant state for one or more groL- strains. It is suggested that a complex between F and rep proteins is involved in phage maturation. The A protein appears to interact with this complex.     

Influence of resistance-factors on the phage types ofSalmonella Panama

The resistance to antibiotics which has been increasingly observed in naturally occurringSalmonella panama, is due to an R-factor. A relationship was found between phage pattern and the presence of this R-factor. All strains belonging to phage types A, C and E are sensitive to all antibiotics and are indicated in phage-typing by wild-type phage 47 or host-range mutants of phage 47. All strains belonging to phage types B, D and F possess an R-factor and are indicated by host-modified variants of phage 47. Phage type G, indicated by a host-range mutant, and group Z contain strains with, as well as without an R-factor. Spontaneous drug-sensitive segregants of type B, D and F strains have the phage pattern A, C and E respectively. Conversely, the phage pattern of A, C and E type strains change into B, D and F respectively after infection with the R-factor ofS. panama. The theory can be advanced that type B type A+R-factor, D — C+R-factor and F = E+R-factor. This change in phage type can be considered to be due to the fact that the R-factor exerts restriction and modification of the phage which indicates theS. panama strain without the R-factor.Many of the antibiotic-resistantEscherichia coli strains found in nature possess an R-factor which can be transferred toS. panama in vitro. Relatively few of these R-factors were found to possess also the restriction marker. Thus up to the present the number ofE. coli strains possessing an R-factor which is able to create a dependable combination of phage type and drug resistance inS. panama is relatively small.