Biosynthetic Origin of Hygromycin A

Hygromycin A, an antibiotic produced by Streptomyces hygroscopicus,is an inhibitor of bacterial ribosomal peptidyl transferase.The antibiotic binds to the ribosome in a distinct but overlappingmanner with other antibiotics and offers a different templatefor generation of new agents effective against multidrug-resistantpathogens. Reported herein are the results from a series ofstable-isotope-incorporation studies demonstrating the biosyntheticorigins of the three distinct structural moieties which comprisehygromycin A. Incorporation of [1-¹³C]mannose and intact incorporationof D-[1,2-¹³C₂]glucose into the 6-deoxy-5-keto-D-arabino-hexofuranosemoiety are consistent with a pathway in which mannose is convertedto an activated L-fucose, via a 4-keto-6-deoxy-D-mannose intermediate,with a subsequent unusual mutation of the pyranose to the correspondingfuranose. The aminocyclitol moiety was labeled by D-[1,2-¹³C₂]glucosein a manner consistent with formation of myo-inositol and asubsequent unprecedented oxidation and transamination of theC-2 hydroxyl group to generate neo-inosamine-2. Incorporationof [carboxy-¹³C]-4-hydroxybenzoic acid and intact incorporationof [2,3-¹³C₂]propionate are consistent with a polyketide synthase-typedecarboxylation condensation to generate the 3,4-dihydroxy- ${alpha}$ -methylcinnamicacid moiety of hygromycin A. No labeling of hygromycin A wasobserved when [3-¹³C]tyrosine, [3-¹³C]phenylalanine, or [carboxy-¹³C]benzoicacid was used, suggesting that the 4-hydroxybenzoic acid isderived directly from chorismic acid. Consistent with this hypothesiswas the observation that hygromycin A titers could be reducedby addition of N-(phosphonomethyl)-glycine (an inhibitor ofchorismic acid biosynthesis) and restored by coaddition of 4-hydroxybenzoicacid. The convergent biosynthetic pathway established for hygromycinA offers significant versatility for applying the techniquesof combinatorial and directed biosynthesis to production ofnew antibiotics which target the ribosomal peptidyl transferaseactivity.

INTRODUCTION

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Introduction

The isolation of hygromycin A (Fig. 1) (14, 19) from severalstrains of Streptomyces hygroscopicus was reported in 1953.A structurally unrelated antibiotic, the aminoglycoside hygromycinB, was later isolated from the same S. hygroscopicus strains(13). Initial studies demonstrated that hygromycin A had a relativelybroad spectrum of modest activity against gram-positive andgram-negative bacteria (14, 19). Hygromycin A has also beenshown recently to have immunosuppressant activity in the mixedpoor lymphocyte reaction but does not work via suppression ofinterleukin 2 production (29).

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FIG. 1. Structures of hygromycin A, antibiotic A201A, and puromycin.

More than 2 decades after its initial isolation the mode ofantibacterial action of hygromycin A was demonstrated to beinhibition of the ribosomal peptidyl transferase activity (7).Hygromycin B also disrupts protein synthesis, but by a differentmechanism, that of inhibition of translocation and misreadingof mRNA (4, 6, 12). Initial studies demonstrated that hygromycinA blocks binding of either radiolabeled chloramphenicol or lincomycinto the ribosome, suggesting that the binding sites of all threeantibiotics are closely related (7). These analyses also demonstratedthat hygromycin A is more potent than chloramphenicol and bindsstrongly to ribosomes (7). More recent competitive footprintingexperiments have shown that the binding of hygromycin A is affectedonly by macrolides containing a mycarose moiety (22). Recentcrystallographic analyses have clearly demonstrated that forthe 16-membered macrolides tylosin and carbomycin, the C-5 disaccharidegroup containing this mycarose extends from the polypeptideexit channel of the large ribosomal subunit into the peptidyltransferase center (8). Taken together, the data clearly implythat hygromycin A binds to the ribosome in this region in adistinct but overlapping manner with other antibiotics. Thedecrease in clinical utility of macrolides due to the appearanceof resistant pathogens has prompted a need to identify agentswhich bind differently to the ribosome. Analogs and derivativesof hygromycin A may offer important possibilities in the developmentof such compounds.

Renewed interest in hygromycin A has also been a result of theobservation that the antibiotic is effective in the treatmentof induced dysentery in pigs at levels of 5 to 20 g/ton of feed(15). Swine dysentery is a severe mucohemorrhagic diarrhealdisease thought to be caused by Serpulina (Treponema) hyodysenteriae(16), an anaerobic spirochete against which hygromycin A hasbeen shown to have good in vitro potency. These observationshave led to the preparation of over 100 analogs and determinationof their activity, in terms both of MICs for S. hyodysenteriaeand of their ability to inhibit protein synthesis in an Escherichiacoli cell-free system (9). This work has been used to constructa structure-activity relationship for hygromycin A, in whichthe aminocyclitol moiety is essential for activity. 6-Deoxy-5-keto-D-arabino-hexofuranoseis not essential for antibacterial activity and can be replacedby a hydrophobic allyl group. This replacement decreases theintrinsic protein synthesis inhibition activity but may allowmore efficient transport into the bacterial cell. The methylgroup in the central 3,4-dihydroxy- ${alpha}$ -methylcinnamic acid moietyis sensitive to change, and replacement with propyl, allyl,or hydrogen substituents leads to a reduction in antibacterialactivity (9, 10). This work has also shown that while hygromycinA is a potent inhibitor of protein synthesis, its ineffectivenessagainst gram-negative enteric bacteria such as E. coli is mainlydue to the efflux mechanism (AcrAB) (9).

The biological activity of hygromycin A and its analogs, coupledwith the observation that it offers ribosomal binding that isdistinct from other antibiotics, prompted us to investigatethe biosynthesis of this antibiotic. This work was undertakenwith a long-term view of generating potentially useful new antibioticlead compounds through techniques such as combinatorial biosynthesisand mutasynthesis. In this paper we detail results of stable-isotope-incorporationstudies which establish the biosynthetic origins of the threemoieties of hygromycin A. The results indicate a convergentbiosynthetic pathway, which suggests significant versatilityfor engineering production of new antibiotics which target thebacterial ribosomal peptidyl transferase activity.

MATERIALS AND METHODS

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Materials and Methods

Cultural conditions for hygromycin A production. Seed cultures of S. hygroscopicus NRRL 2388 were prepared byinoculating spores into a 500-ml baffled flask containing 50ml of medium consisting of 13 g of glucose, 7.0 g of corn starch,7.0 g of NZ amine YTT (casein hydrolysate; Quest InternationalFlavors, Norwich, N.Y.), 3.0 g of corn steep solid (spray-driedcorn steep liquor; Roquette Corp., Gurnee, Ill.), 1.3 g of MgSO₄· 7H₂O, 0.7 g of (NH₄)₂PO₄, 0.7 g of KH₂PO₄, 2 mg ofCoCl₂ · 6H₂O, 2 drops of soybean oil, and 1 drop of P2000(polypropylene glycol; Fluka) in 1 liter of tap water, pH 7.0.This seed culture was incubated for 2 days at 30°C and 280rpm and used to provide a 4% inoculum for 100 ml of productionmedium consisting of 50 g of glucose, 10 g of soy flour, 5.0g of NZ amine YTT, 5.0 g of NaCl, 1.0 g of CaCO₃, 2 drops ofsoybean oil, and 1 drop of P2000 in 1 liter of tap water (pH7.0). This hygromycin A production culture was then incubatedin a 500-ml baffled flask for 6 days at 30°C and 280 rpm.

Hygromycin A purification. The culture filtrate was adsorbed by 1/20 volume of AmberliteXAD-4 (Sigma) at 5°C for 2 h. The column was washed withwater and then eluted with methanol. The pooled fractions containinghygromycin A were further purified by preparative high-pressureliquid chromatography with a C₁₈ reverse-phase column (250 by10.0 mm; Phenomenex, Torrance, Calif.) using 20% acetonitrile-80%water as the eluent (2 ml/min). Hygromycin A was detected bymonitoring at 272 nm with a UV detector. The hygromycin A eluatewas freeze-dried and analyzed by ¹H and ¹³C nuclear magneticresonance (NMR) analyses (using previously published ¹³C assignments[33]). As previously observed (10, 33), the hygromycin A obtainedfrom the fermentations typically contained a minor C-4" epihygromycin(Fig. 1) for which only the ¹³C resonances associated with thefuranoside moiety were different.

Incorporation of isotope-labeled precursors into hygromycin A. The labeled compounds D-[1,2-¹³C₂]glucose, D-[1-¹³C]mannose,L-[methyl-¹³C]methionine, sodium [2,3-¹³C₂]propionate, and [3-¹³C]tyrosinewere all obtained from Cambridge Isotopes Laboratories, Inc.[3-¹³C]phenylalanine was obtained from CDN Isotopes Inc. [carboxy-¹³C]4-hydroxybenzoicacid and [carboxy-¹³C]benzoic acid were obtained from Isotec,Inc. All compounds were 99% labeled and separately pulse fedin four equal batches to a 100-ml culture of a hygromycin Aproducing culture of S. hygroscopicus at 3, 3.5, 4, and 4.5days. The final concentration of the labeled materials addedwas 0.6 mg/ml of the production medium. After a total of 6 daysof fermentation, hygromycin A was purified as described above.Approximately 25 to 50 mg of purified hygromycin A was obtainedfrom each separate incorporation experiment. The ¹³C NMR spectrumof purified labeled hygromycin A in CD₃OD was recorded on a300 MHz NMR spectrometer (Varian Inc., Palo Alto, Calif.) andcompared with standard hygromycin A provided by Pfizer and analyzedunder the same conditions.

Inhibition of hygromycin A biosynthesis. N-(Phosphonomethyl)-glycine was added to the production mediumat 24 h of fermentation at a final concentration of 0.5 mg/ml.In another experiment, same concentration of N-(phosphonomethyl)-glycineand 0.25 mg of 4-hydroxybenzoic acid (Aldrich) per ml were addedto the production medium at 24 h of fermentation. A controlexperiment was carried out at the same time. All fermentationswere carried out in duplicate, and a time course of hygromycinA was determined by using a high-pressure liquid chromatographyassay.

¹³C NMR analyses. A region selective sensitivity enhanced heteronuclear singlequantum coherence (HSQC) experiment (30) was performed withthe hygromycin A sample from the D-[1,2-¹³C₂]glucose incorporationexperiment. This experiment allows observation of ¹³C-¹³C scalarcoupling in the indirectly detected dimension and offers significantsensitivity advantages over ¹³C-¹³C INADEQUATE experiments anddirect observation of ¹³C scalar couplings in conventional one-dimensional¹³C experiments. The data set was obtained on a Varian Unity+500 MHz spectrometer equipped with a quadruple resonance probeand consisted of 1,024 complex t₁ increments. Each t₁ incrementconsisted of 1,024 complex points averaged for 16 transientswith a 1.7-s repetition rate. Data were processed for phase-sensitivedisplay with FELIX v97.2 (Accelrys, San Diego, Calif.) usingcosine bell weighting functions in both dimensions.

RESULTS AND DISCUSSION

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Results and Discussion

Hygromycin A production time course. In order to determine the best time to add ¹³C-labeled biosyntheticprecursors, we established a time course for hygromycin A productionunder our laboratory fermentation conditions (data not shown).This analysis showed that during the exponential growth phase(0 to 48 h), a low level of hygromycin A was produced. HygromycinA production continued during the stationary phase and reacheda maximal level of approximately 700 µg/ml by day 6. Basedon these results, we chose to add biosynthetic precursors ina batchwise process between 72 and 108 h, during the periodwhen the rate of hygromycin production was maximal.

Biosynthesis of the 6-deoxy-5-keto-D-arabino-hexofuranose moiety. The stereochemical configuration at C-2"of hygromycin A indicatesthat mannose rather than glucose is the more immediate precursorof the 6-deoxy-5-keto-D-arabino-hexofuranose-derived moiety(Fig. 2). To test this hypothesis, [1-¹³C]mannose was fed toproducing cultures. The resulting hygromycin A (34 mg) was shownby NMR analyses to be specifically enriched (threefold) at C-1"(103.7 ppm). In this experiment we were unable to detect anylabeling of the aminocyclitol moiety of hygromycin A from mannose.As the aminocyclitol is efficiently labeled from D-[1,2-¹³C₂]glucose(see below), this result indicates that under the conditionsof this experiment the carbon flux is from glucose towards mannose.Consistent with this hypothesis was the efficient intact labelingof the 6-deoxy-5-keto-D-arabino-hexofuranose-derived moietyfrom glucose. In our incorporation study with D-[1,2-¹³C₂]glucose,analyses of cross-peak volumes in the HSQC revealed a clearand unambiguous 1% intact labeling of hygromycin A (40 mg) (J= 50 Hz) at C-1" (103.7 ppm) and C-2" (78.6 ppm) (Fig. 3c).This labeling study also clearly demonstrated that the publishedassignments (33) for C-2" (78.2 ppm) and C-5 (78.6 ppm) shouldbe reversed. Intact labeling was also observed for the C-1"and C-2" signals associated with epihygromycin A. No enrichmentsof other positions in the furanose moiety were observed.

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FIG. 2. Proposed pathway for production of the 6-deoxy-5-keto-D-arabino-hexofuranose and aminocyclitol moieties of hygromycin A from glucose. The bold bonds indicate intact labeling of hygromycin A from D-[1,2-¹³C₂]glucose (low-level intact labeling of C-3 and C-4 of hygromycin was also observed). The "13" indicates labeling of the furanose ring of hygromycin A by [1-¹³C]mannose.

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FIG. 3. ¹³C NMR analyses revealing efficient intact labeling of C-1-C-2 (A) and C-1"-C-2" (C) and lower intact labeling of C-3-C-4 (B) of hygromycin A from D-[1,2-¹³C₂]glucose. Spectra are one-dimensional ¹³C slices from the HSQC data set for the hygromycin A sample from the D-[1,2-¹³C₂]glucose labeling experiment.

These results are consistent with a pathway in which D-glucose-6-phosphateis converted via D-fructose-6 phosphate to D-mannose-1-phosphate(Fig. 2). In subsequent steps, this intermediate is likely convertedto a nucleoside diphosphate (NDP)-activated L-fucose. L-Fucoseis found in glycoconjugates of plants, animals, and bacteria(26, 31), including streptomycetes (34), and plays a role inmaintaining structural integrity and molecular recognition.This pathway would involve elimination of water to generatean NDP-4-keto-6-deoxy-D-mannose, a step catalyzed by an enzymesuch as GDP-D-mannose 4,6-dehydratase. This intermediate wouldthen be converted to GDP-L-fucose by GDP-L-fucose synthetase,which catalyzes epimerization at the C-3 and C-5 positions ofthe hexose ring, and an NADPH-dependent reduction at the C-4position (11, 26). It is unclear whether any of the genes requiredfor this formation of the proposed NDP-activated L-fucose arelocated within the hygromycin A biosynthetic gene cluster. Itis worth noting that genes encoding enzymes with homology toGDP-D-mannose 4,6-dehydratase have recently been identifiedin the nystatin, candicidin, and antibiotic A201A biosyntheticgene clusters (3, 5, 24). A mutation of the NDP-L-fucose (apyranose) to the furanose and oxidation of the 5-keto groupwould provide the activated 6-deoxy-5-keto-D-arabino-hexofuranosefor attachment to the C-4 hydroxyl of the 3,4-dihydroxy- ${alpha}$ -methylcinnamicacid-derived moiety of hygromycin A. The mutase responsiblefor the ring contraction of GDP-L-fucose likely follows themechanism recently established for the primary metabolic UDP-galactopyranomutase(23, 35). To the best of our knowledge, no rearrangements ofthis type have been reported previously for antibiotic-biosyntheticpathways. In this regard we note that antibiotic A201A, whichhas significant structural similarities to hygromycin A, containsa moiety that is likely derived from 5-keto-D-arabino-hexofuranose(Fig. 1). Biosynthetic experiments have not been reported forantibiotic A201A, and a partial sequence of the biosyntheticgene cluster (24) has not yet identified genes involved in formationof the furanose moiety.

Biosynthesis of the aminocyclitol moiety. It has been proposed that the unusual neo-inosamine-2 of hygromycinA is presumably formed via a pathway which involves myo-inositolas an intermediate (18). This pathway has been studied for thescyllo-inosamine-derived moiety of streptomycin (1). In thelatter case, oxidation of the C-2 hydroxyl of myo-inositol (derivedfrom C-5 of glucose) and subsequent transamination (catalyzedby L-glutamine:scyllo-inosose amino transferase) (1) yieldsscyllo-inosamine. A similar process occurring at C-5 of myo-inositol(derived from C-2 of glucose) would be predicted to generateneo-inosamine-2 (Fig. 2). To test this possibility, we carriedout an incorporation study with D-[1,2-¹³C₂]glucose. Consistentwith our predictions, the HSQC analysis of the hygromycin Aobtained from this study demonstrated clear and unequivocalintact labeling at C-1 (71.6 ppm) (Fig. 3A) and C-2 (50.7 ppm)(J = 39.5 Hz) (0.7%). There was no detectable labeling at C-5or C-6, consistent with divergent pathways leading from myo-inositolto neo-inosamine-2 and scyllo-inosamine. However, a low levelof intact labeling (J = 44.8 Hz) (0.2%) at C-3 (72.6 ppm) (Fig.3B) and C-4 (78.2 ppm) of hygromycin A was observed. This observationsuggests that an alternative process or pathway must contribute,albeit to a lesser degree, to formation of neo-inosamine-2 fromglucose.

The C-4 and C-5 hydroxyl groups in hygromycin A are bridgedby a methylene group. We hypothesized that S-adenosylmethioninewas the source of this methylene group and carried out an incorporationstudy with L-[methyl-¹³C]methionine. Consistent with our predictions,the ¹³C NMR analysis of the resulting hygromycin A (50 mg) exhibiteda single 25-fold enrichment at 96.2 ppm, the resonance assignedto the methylene bridge. It remains to be determined at whichstage during the biosynthetic process the methylene group isintroduced onto the aminocyclitol ring. The high level of enrichmentof hygromycin A by methionine in this experiment suggested thatunder our fermentation conditions methionine may be a limitingfactor in fermentation titers. While a comprehensive fermentationstudy is needed to test this hypothesis, it is interesting thatthe highest level of hygromycin A in this series of studieswas obtained in the methionine feeding study.

Biosynthesis of the 3,4-dihydroxy- ${alpha}$ -methylcinnamic acid moiety. Hygromycin A, antibiotic A201A, and puromycin are all inhibitorsof protein synthesis (albeit with specific differences in theirmechanisms of action) and have been observed to have some similaritiesin structure (Fig. 1) (10). In particular, the 3,4-dihydroxy- ${alpha}$ -methylcinnamicacid moiety of hygromycin A and the 4-hydroxy- ${alpha}$ -methylcinnamicacid of antibiotic A201A are similar to the tyrosine-derivedmoiety of puromycin A (28). Despite these similarities, it isclear that a more complex process must provide the moietiesof hygromycin A and antibiotic A201A. No incorporation at theC-2' CH₃ residue of hygromycin A was observed in the methionineincorporation experiment, ruling out an S-adenosylmethionine-dependentmethylation process. Thus, we hypothesized that in the caseof hygromycin A and antibiotic A201A, these moieties are derivedfrom a 4-hydroxybenzoyl coenzyme A (CoA) or 3,4-dihydroxybenzoyl-CoAstarter unit which is elongated by a methylmalonyl-CoA-derivedextender unit (Fig. 4). Support for this hypothesis was obtainedin an incorporation experiment with [2,3-¹³C₂]propionate, where¹³C NMR analysis of the resulting hygromycin A (31 mg) revealeda specific 1% intact incorporation (J = 44.1 Hz) of the two¹³C labels at the predicted sites, C-2' (132.2 ppm) and theC-2' CH₃ (14.6 ppm).

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FIG. 4. Proposed pathway for the production of the 3,4-dihydroxy- ${alpha}$ -methylcinnamic acid moiety of hygromycin A. The bold bonds indicate intact labeling of hygromycin A from [2,3-¹³C₂]propionate. The "13" indicates labeling of the hygromycin A by [carboxy-¹³C]4-hydroxybenzoic acid. Wavy lines indicate no detectable incorporation of ¹³C label from phenylalanine, tyrosine, and benzoic acid. SR is a CoA or acyl carrier protein thioester.

We hypothesized that the remainder of the 3,4-dihydroxy- ${alpha}$ -methylcinnamicacid moiety may be derived from degradation of phenylalanineor tyrosine. It has recently been established that the benzoyl-CoAstarter unit for the biosynthesis of the polyketide enterocinin Streptomyces maritimus is derived from ß-oxidationof trans-cinnamic acid (derived from phenylalanine by a phenylammonia lyase) (17, 32). Certain bacteria can form benzoyl-CoAvia successive rounds of ${alpha}$ -oxidative decarboxylations of phenylpyruvate(formed by transamination of phenylalanine) (25). Recent evidencehas also shown that the 3-dimethylallyl-4-hydroxybenzoic acidmoiety of clorobiocin is formed from phenylalanine in a processthat involves a prenylation and a retro-aldol reaction (20).We carried out two separate incorporation experiments with [3-¹³C]tyrosineand [3-¹³C]phenylalanine and obtained 31 and 20 mg of hygromycinA, respectively. In neither case was there any detectable levelof ¹³C enrichment at the predicted C-3' (135.0 ppm) of hygromycinA. However, we did observe a strong specific sixfold enrichmentat this carbon when we analyzed hygromycin A (25 mg) obtainedfrom an incorporation study using [carboxy-¹³C]-4-hydroxybenzoicacid. When this experiment was repeated with [carboxy-¹³C]benzoicacid, analysis of the hygromycin A (15 mg) by ¹³C NMR revealedno enrichment at C-3'. These analyses suggest that it is 4-hydroxybenzoyl-CoAwhich is coupled with a methylmalonyl-CoA to generate the 3,4-dihydroxy- ${alpha}$ -methylcinnamicacid moiety of hygromycin A. The addition of the 3-hydroxylgroup may occur prior to or after this elongation step. Thedata also suggest that neither hydroxylation of benzoyl-CoAnor degradation of tyrosine or phenylalanine plays a significantrole in the formation of 4-hydroxybenzoyl-CoA. In bacteria,4-hydroxybenzoic acid is derived from the shikimic acid pathwayintermediate chorismic acid by the action of chorismate lyase(2, 21). This 4-hydroxybenzoic acid is then converted to ubiquinoneby a series of steps, including decarboxylation and prenylation.Activation of this ubiquinone biosynthetic pathway intermediate,4-hydroxybenzoic acid, to the appropriate thioester could providethe appropriate precursor for hygromycin A biosynthesis (Fig.4).

The formation of the 3,4-dihydroxy- ${alpha}$ -methylcinnamic acid moietyof hygromycin A most likely is catalyzed by a polyketide synthase.In this process, the 3-keto group of the product, generatedby condensation of the methylmalonyl-CoA and either 4-hydroxybenzoyl-CoAor 3,4-dihydroxybenzoyl-CoA, would be processed by reductionand dehydration. Consistent with our prediction is a findingin a recent analysis of the portion of the antibiotic A201Agene cluster of the gene ataPKS1, which encodes a protein withsome homology to the acyltransferase domains of modular typeI polyketide synthases (24).

Inhibition of hygromycin A biosynthesis by N-(phosphonomethyl)-glycine. Our incorporation studies indicate that the 4-hydroxybenzoicacid-derived moiety of hygromycin A is derived from the shikimicacid pathway intermediate chorismic acid. To test this hypothesis,we carried out a fermentation in the presence of N-(phosphonomethyl)-glycine,an inhibitor of 5-enol-pyruvylshikimic acid 3-phosphate synthase(27), which catalyzes a key step in chorismic acid biosynthesis.The inhibitor was added to the production medium after 24 hof fermentation, allowing the cells to grow and thus avoidinginhibitory effects on ubiquinone biosynthesis. Under these conditionswe saw normal cell growth but 20 and 40% decreases in the titersof hygromycin A for inhibitor concentrations of 20 and 50 mg/100ml, respectively. Higher inhibitor concentrations did not yieldadditional decreases in fermentation titers. A complete lossof hygromycin A was not expected in these experiments. Our timecourse for hygromycin A production indicated that approximately15% of the overall hygromycin A is generated by 24 h of fermentation(prior to addition of the inhibitor), and there is likely tobe some 4-hydroxybenzoic acid available to support additionalhygromycin A biosynthesis.

We also carried out a fermentation time course experiment todetermine if addition of 4-hydroxybenzoic acid can restore hygromycinA to cultures treated with N-(phosphonomethyl)-glycine. As shownin Fig. 5, production of hygromycin A in the control continuedthrough day 6, whereas in the cultures treated with the inhibitor,production stopped essentially by day 3. Continued productionof hygromycin A through day 7, with levels comparable to thecontrol, were observed when cultures were treated with a mixtureof the inhibitor and 4-hydroxybenzoic acid. This series of experimentstogether with the labeling experiments described above are allconsistent with the proposed pathway to the 4-hydroxybenzoyl-CoAprecursor for hygromycin A biosynthesis (Fig. 4).

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FIG. 5. Inhibition of hygromycin A production by N-(phosphonomethyl)-glycine can be overcome by addition of exogenous 4-hydroxybenzoic acid. Circles, control fermentation; squares, addition of inhibitor at 24 h; triangles, addition of inhibitor and exogenous 4-hydroxybenzoic acid at 24 h. See the text for details.

Summary. This study has clearly established the biosynthetic originsof hygromycin A from mannose, glucose, 4-hydroxybenzoic acid,propionate, and methionine. The incorporation results indicatethat there are unique features for each of the pathways providingthe three moieties of hygromycin A. These moieties are likelyassembled separately and then joined by using an amide synthaseand glycosyl transferase. Such a convergent biosynthetic routeoffers tremendous potential for structural modification viatechniques of mutasynthesis and combinatorial biosynthesis.The first step in this process, the identification, cloning,and sequencing of the hygromycin A gene cluster, is under wayin our laboratory, using appropriate probes designed based onknowledge gained through the biosynthetic studies describedherein.

ACKNOWLEDGMENTS

We thank Hamish McArthur and Maria Brown (Pfizer Incorporated,Central Research Division) for providing fiscal support.

We thank Hamish McArthur and Maria Brown for details on growthof S. hygroscopicus (NRRL 2388) and isolation and purificationof hygromycin A. We also thank Ben Liu (University of Texas,Austin) for helpful discussions on the biosynthetic originsof the 6-deoxy-5-keto-D-arabino-hexofuranose moiety of hygromycinA.

FOOTNOTES

* Corresponding author. Mailing address: Institute for Structural Biology and Drug Discovery, Suite 212B, Biotechnology Park, 800 East Leigh St., Richmond, VA 23219. Phone: (804) 828-7575. Fax: (804) 827-3664. E-mail: kareynol{at}hsc.vcu.edu.

Permanent address: Department of Microbiology, Faculty of Pharmacy,Mansoura University, Mansoura, Egypt.

REFERENCES

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