Control ofAntibiotic Biosynthesis · roides, whicharenovobiocinproducers (4, 72). The...

MICROBIOLOGICAL REVIEWS, June 1980, p. 230-251 Vol. 44, No. 20146-0749/80/02-0230/22$02.00/0

Control of Antibiotic BiosynthesisJUAN F. MARTIN' AND ARNOLD L. DEMAIN2

Department ofMicrobiology, Faculty ofPharmacy, University of Salamanca, Salamanca, Spain' andDepartment of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge,

Massachusetts 021392

INTRODUCTION: PRIMARY AND SECONDARY METABOLISM .............. 230LATE FORMATION OF ANTIBIOTICS IN BATCH CULTURE: GROWTH AND

PRODUCTION PHASES .................................................. 231WHY IS THE BIOSYNTHESIS OF ANTIBIOTICS OFTEN DELAYED UNTIL

THE IDIOPHASE?-SURVIVAL IMPLICATIONS ......................... 232CONTROL OF INITIATION OF ANTIBIOTIC SYNTHESIS: ENZYME REPRES-

SION AND INHIBITION .................................................. 232Repression of Antibiotic Synthetases ....................................... 232Inhibition of Antibiotic Synthetases 234Inhibition in a Branched Pathway Leading to a Primary Metabolite and anAntibiotic 234

INTRACELLULAR EFFECTORS AND MECHANISMS THAT CONTROL THEONSET OF ANTIBIOTIC SYNTHESIS ..................................... 235

Carbon Catabolite Regulation ............................................ 235Nitrogen Metabolite Regulation 236Phosphate Regulation .............. 237Induction 239

CESSATION OF ANTIBIOTIC BIOSYNTHESIS 241Irreversible Decay of Antibiotic Synthetases ............ 242Feedback Regulation by Antibiotics of Their Own Biosynthesis 242

GENETIC MANIPULATION OF THE MECHANISMS CONTROLLING ANTI-BIOTIC BIOSYNTHESIS ...243

SUMMARY 244LITERATURE CITED 244

INTRODUCTION: PRIMARY ANDSECONDARY METABOLISM

The concept of secondary metabolism as pro-posed initially (16, 17, 195, 196) was oversimpli-fied, but it is still useful for understanding thebiosynthesis of microbial metabolites. Primaryand secondary metabolites might be bettertermed "general" and "special" metabolites, re-

spectively (129). Primary metabolism involvesan interconnected series of enzyme-mediatedcatabolic, amphibolic, and anabolic pathways,which provide biosynthetic intermediates andenergy and convert biosynthetic precursors intoessential macromolecules, such as deoxyribonu-cleic acid, ribonucleic acid, protein, lipids, andpolysaccharides. Primary metabolism is essen-tially identical for all living things. The meta-bolic reactions of primary metabolism are finelybalanced, and metabolic intermediates otherthan those necessary for cell survival rarely ac-cumulate. In addition to this general type ofmetabolism, certain taxonomic groups are ca-pable ofsynthesizing special types of metabolitesby using either the same general enzymes orspecial synthetases produced by specific cellsunder specific nutritional conditions. Thus, fatty

acid synthesis is common to all living cells,whereas polyketide synthesis, in which the sameprecursors and similar biosynthetic enzymes areused, is restricted to certain taxonomic groupsof microorganisms and plants.Secondary metabolites have also been termed

"idiolites" (188) because they are formed duringthe idiophase (production phase) of batch cul-tures (see below). These special metabolites usu-ally possess bizarre chemical structures and arenot essential for growth of the producing orga-nism, although they probably have survivalfunctions in nature. Secondary metabolites areproduced only by some species of a genus, usu-ally as families of closely related components.The chemical diversity and unusual structures

of idiolites are illustrated by the many classes oforganic compounds to which they belong. Theseinclude, among many others, amino sugars, qui-nones, coumarins, epoxides, ergot alkaloids, glu-tarimides, glycosides, indole derivatives, lac-tones, macrolides, naphthalenes, nucleosides,peptides, phenazines, polyacetylenes, polyenes,pyrroles, quinolines, terpenoids, and tetracy-clines (12, 195). Secondary metabolites includeunusual chemical linkages, such as ,B-lactamrings, cyclic peptides made of normal and mod-

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CONTROL OF ANTIBIOTIC BIOSYNTHESIS 231

ified amino acids, unsaturated bonds of polyace-tylenes and polyenes, and large rings of macro-lides. Idiolites are produced typically as mem-bers of a particular chemical family. There areat least 10 natural penicillins, 3 neomycins, 4tyrocidines, 5 mitomycins, 10 bacitracins, 10polymyxins, 20 actinomycins, 4 levorins, 4 poly-fungins, and 13 bleomycins. One Micromono-spora strain produces no fewer than 48 amino-cycitol antibiotics (12). The recent developmentof rapid separation and analytical techniqueshas increased markedly the number of minorcomponents isolated.The proportion of each component in a mix-

ture depends on genetic and environmental fac-tors, apparently because of the low specificity ofthe enzymes involved in secondary metabolism.In contrast, in primary metabolism the biosyn-thetic processes are always carried out withgreat specificity; generally, only one substrate isaccepted and only one product is formed. Pri-mary metabolism has narrow specificity becauseerrors in the biosynthesis of essential cell com-ponents are usually lethal, whereas errors insecondary metabolism are usually of no conse-quence to the producing cells, especially sincethe modified metabolite often retains biologicalactivity.Secondary metabolites are synthesized by a

greater variety of pathways than are primarymetabolites. Despite this variety of pathwaysand final products, most idiolites are assembledfrom a few key intermediary metabolites.An important characteristic of secondary me-

tabolism is that idiolites are usually producedonly at low specific growth rates ofthe producingcultures. This type of regulation affects a wholerange of biosynthetic processes. Individual bio-synthetic pathways are also affected by regula-tory mechanisms, such as induction, cataboliteregulation, and end product regulation. In otherwords, secondary metabolism seems to be gov-erned by (i) overall regulatory controls whichoperate as functions of growth rate and (ii) spe-cific regulatory effects on individual pathways(129).Although the rest of this paper deals with

antibiotics, we feel that most of the conceptsdiscussed are applicable to non-antibiotic sec-ondary metabolites, such as pigments, toxins,and plant growth factors.

LATE FORMATION OF ANTIBIOTICS INBATCH CULTURE: GROWTH AND

PRODUCTION PHASESIn batch cultures containing nutritionally rich

media, high levels of antibiotics are usually pro-duced only after most of the cellular growth hasalready occurred (181, 195). This fact is clearly

observed in antibiotic-producing unicelluar bac-terial cultures with well-defined exponentialgrowth phases (9, 13, 52, 80, 108, 109, 155, 171).The growth phase is called the "trophophase,"whereas the production phase is termed the"idiophase" (20).The separation between trophophase and idi-

ophase is not so clear-cut in filamentous micro-organisms (actinomycetes and fungi). In manyfernentations of filamentous microorganisms,dry cell weight continues to increase signifi-cantly during the idiophase, although at a lowerrate than earlier in the trophophase. However,dry weight is a poor criterion of true growth.Cellular mass consists of true structural materialrequired for cell replication (cell walls, mem-branes, cytoplasmic organelles, ribosomes, nu-clei, etc.) and assimilatory reserve materials,such as polyols, lipids, polyphosphates, andnonstructural carbohydrates. Nonreplicatorygrowth usually results from the accumulation ofreserve materials, which may account for up to50 to 60% of the dry weight at the end of afermentation. Probably the best parameter withwhich to measure true replicatory growth is theincrease in deoxyribonucleic acid (137). In thiscase, cell growth often can be clearly dissociatedfrom antibiotic production (Fig. 1). Other pa-rameters that often indicate the end of the rep-licatory growth phase are a drop in respiratoryactivity (137) and a decrease in the ribonucleicacid synthesis rate (88, 89, 113, 143).

In many antibiotic fermentations (e.g., chlor-amphenicol, colistin, penicillin, and bacitracin),typical trophophase-idiophase dynamics occurin complex media capable of supporting rapidgrowth, but the two phases overlap in definedmedia supporting slow growth (50, 66-68, 84,

1200k

E

_~ 800

qua,

o

80

z40

20

O

TIME (hours)

FIG. 1. Deoxyribonucleic acid (DNA) synthesisand increase in dry cell weight of S. griseus in rela-tion to candicidin production. Redrawn from refer-ence 137.

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232 MARTIN AND DEMAIN

118, 123, 158, 177). The factor that controls theonset of antibiotic biosynthesis is probably thedeficiency of one or more nutritional growth-limiting components. Depletion of such a factorarrests growth and initiates idiolite biosynthesisby mechanisms which are only beginning to beunderstood (see below). In defined medium sup-

porting only slow growth, some nutritional factormay be growth limiting from the very start ofcultivation, thus favoring antibiotic productionwhile slow growth is still occurring.We emphasize that the timing of product for-

mation should not be used to define a secondarymetabolite. As discussed above, trophophaseand idiophase ideally occur at two separate timesin batch cultures, but often they overlap. Asecondary metabolite is secondary only becauseit is not necessary for exponential growth of theproducing culture.

WHY IS THE BIOSYNTHESIS OFANTIBIOTICS OFTEN DELAYED UNTIL

THE IDIOPHASE?-SURVIVALIMPLICATIONS

Microorganisms appear to be programmed toproduce antibiotics only when the specificgrowth rate decreases below a certain level (19).This phenomenon probably was established dur-ing evolution in response to competitive pres-sures. We believe that one role of antibiotics innature is to inhibit or kill other microorganisms.Research on this controversial point has beenreviewed by Gottlieb (60). Our own view on thissubject has been discussed by Katz and Demain(91).In nutritionally rich habitats, such as the in-

testines of mammals, antibiotic production bymicroorganisms is not necessary, since enoughfood is available for all species present. However,in most natural habitats (e.g., soil and water),nutrients are limiting for microbial growth, andantibiotic production is advantageous for sur-vival. A survival advantage has been clearlydemonstrated in Cephalosporium gramineum(15). Nutrient deficiency often induces differen-tiation (e.g., endospore formation in bacilli andconidiation in streptomycetes), and some anti-biotics may act as differentiation effectors (32,71, 168, 171); i.e., they may be necessary for theformation of spores or for proper timing of sporegermination. If antibiotics do effect differentia-tion, their biosynthesis should be limited to theperiod of the differentiation process that re-

quires their action. Experimental evidenceshows that antibiotic production in bacilli is anearly event, occurring usually at stage 0 or I inthe sporulation process.

Antibiotic production only after the growth

phase nears completion is useful to the produc-ing strain, since antibiotic-producing species aresensitive during growth to the antibiotic thatthey produce. Usually this sensitivity is re-stricted to the growth phase before antibioticproduction, and only later does the producingstrain become resistant to its antibiotic. Manyreports illustrate this phenomenon. Growthfrom a spore inoculum of Streptomyces griseo-carneus, a hydroxystreptomycin producer, is in-hibited by 5 ,tg of streptomycin per ml, whereas300 ,ug/ml added to 24-h mycelia has no effect(11). Growth of Streptomyces antibioticus, anactinomycin producer, is inhibited 50% by 4 ,ugof actinomycin per ml, although the strain iscapable of producing about 120 ,ug/ml of idi-ophase. Similar results have been reported inStreptomyces parvulus, another actinomycinproducer (70). Early growth of the tetracyclineproducer Streptomyces aureofaciens is inhibitedby 400 ,ug of chlortetracycline per ml, eventhough protein synthesis occurs during the lateidiophase in the presence of 2,000 ,ug/ml (144).Similarly, chloramphenicol inhibits the growthof its producing strain, Streptomyces sp. 3022a(124), and novobiocin inhibits growth of bothStreptomyces niveus and Streptomyces sphe-roides, which are novobiocin producers (4, 72).The antibiotic-producing species manage to

avoid suicide (27) by mechanisms that include(i) modification (and thus detoxification) of theantibiotic by enzymes formed by the antibiotic-producing strain, (ii) alteration of the antibiotictarget in the producing cell, and (iii) a decreasedinward permeability to the antibiotic after it hasbeen excreted.

CONTROL OF INITIATION OFANTIBIOTIC SYNTHESIS: ENZYMEREPRESSION AND HIBMITION

The genes containing the genetic informationfor antibiotic biosynthesis are both chromo-somal and extrachromosomal (73). In at leastone case, structural genes for antibiotic synthesishave been located on plasmids. More frequently,the structural genes are chromosomal, whereasregulatory genes controlling the expression ofthe genetic information appear to be extrachro-mosomal (73).

Expression of the genes coding for antibioticbiosynthesis usually does not occur at highgrowth rates. This phenomenon suggests thatduring rapid growth either antibiotic synthe-tases are not formed or, if formed, their activityis inhibited.

Repression of Antibiotic SynthetasesMany investigators have closely monitored

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synthesis of an antibiotic synthetase and ob-served late enzyme formation. Such control ofgene expression could occur by interference withtranscription of genetic information from deoxy-ribonucleic acid to messenger ribonucleic acid or

by interference with translation of informationfrom messenger ribonucleic acid to the antibioticsynthetase. We do not know exactly at whichlevel the repression control is exerted in thecases of streptomycin, tylosin, actinomycin, pyr-

rolopyrimidine nucleosides, gramicidin S, andtyrocidine, which are described below.

Studies on streptomycin biosynthesis byWalker and co-workers (187, 189, 191, 193) re-

vealed at least two enzymes, amidinotransferaseand streptidine kinase, that are repressed duringthe growth phase. Amidinotransferase (L-argi-nine:inosamine-phosphate amidinotransferase)is involved in the biosynthesis of the streptidinemoiety of streptomycin; it catalyzes two reac-

tions having the following pathway:

O-phosphorylinosamine + arginine -+O-phosphoryl-N-amidinoinosamine

+ ornithine

O-phosphoryl-N-amidinostreptamine+ arginine --+O-phosphorylstreptidine

+ ornithine

There is controversy about whether these tworeactions are catalyzed by two different amidi-notransferases (159, 190). The amidinotransfer-ase(s) is found only in Streptomyces griseus,Streptomyces humidus, S. griseocarneus, Strep-tomyces galbus, Streptomyces ornatus, andStreptomyces hygroscopicus forma glebosus,which all produce streptomycin or the closelyrelated antibiotics dihydrostreptomycin andbluensomycin (95, 153, 156, 188, 191). This im-portant enzyme is formed before the onset ofstreptomycin production but not during growth.The enzyme appears as a result of de novo

protein synthesis, since the process can be in-hibited by chloramphenicol.The enzyme streptidine kinase phosphoryl-

ates streptidine by the following reaction:

streptidine + adenosine triphosphateO-phosphorylstreptidine

+ adenosine diphosphate

Its activity is very low during trophophase andmarkedly increases during idiophase (191).The sugars and amino sugars of the amino-

cycitol and macrolide antibiotics are synthe-sized from glucose via nucleoside diphosphate-sugar derivatives. Thymidine diphosphate-4-keto-6-deoxyglucose is an intermediate in theformation of thymidine diphosphate-mycarose

from thymidine diphosphate-glucose (151). De-oxythymidine diphosphate4-keto-6-deoxyglu-cose is an intermediate in the biosynthesis ofdeoxythymidine diphosphate-dihydrostreptose,a precursor of streptomycin (150). Thymidinediphosphate4-keto-6-deoxyglucose is formedby a nicotinamide adenine dinucleotide phos-phate-dependent thymidine diphosphate-glu-cose oxidoreductase. The activity of this oxido-reductase in Streptomyces rimosus, which pro-duces the macrolide antibiotic tylosin, increasesduring the stationary phase before production oftylosin (140). Conversion of thymidine diphos-phate-4-keto-6-deoxyglucose to thymidine di-phosphate-L-mycarose involves transmethyla-tion and reduction of the 4-keto group. Thetransmethylase of S. rimosus is also formed afterthe growth phase (140).The actinomycin molecule is formed from two

molecules of4-methyl-3-hydroxyanthranilic acidpentapeptide by the action of the enzyme phen-oxazinone synthase (90). This enzyme is a typicalidiophase enzyme specific for actinomycin pro-ducers (and organisms that synthesize relatedactinocin-like compounds). There is little syn-thesis ofphenoxazinone synthase during the first20 h of fermentation. Between 20 and 36 h, itsspecific activity increases 5- to 6-fold, and by 48h its specific activity increases 12-fold. Detecta-ble amounts of actinomycin are present onlyafter 24 h, lagging behind the enzyme synthesis(54). Addition of chloramphenicol or puromycinbefore the enzyme is formed inhibits both itsappearance and actinomycin synthesis. Lateraddition of chloramphenicol prevents furtherphenoxazinone synthase increases but not acti-nomycin production by the enzymes alreadyformed (92, 126).

S. rimosus produces the pyrrolopyrimidinenucleoside antibiotics toyocamycin and sangi-vamycin. The pyrrole ring is derived from gua-nosine triphosphate via the action of guanosinetriphosphate-8-formylhydrolase (44, 45). Thisenzyme is specific for production of pyrrolopy-rimidine nucleoside antibiotics; i.e., it is presentin Streptomyces sparsogensis, the producer oftubercidin (a pyrrolopyrimidine nucleoside), butit is not found in Streptomyces showdoensis, aproducer of showdomycin (a nucleoside anti-biotic which is not a member of this group). The8-formylhydrolase, which catalyzes the releaseof carbon 8 of the imidazole ring of guanosinetriphosphate as formic acid, rapidly increases inspecific activity at the end of the growth phaseand precedes sangivamycin production by a fewhours. If added at the end of trophophase, chlor-amphenicol completely inhibits further enzymeproduction and inhibits sangivamycin produc-tion by 92%.

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Gramicidin S synthetase, tyrocidine synthe-tase, and bacitracin synthetase, which are in-volved in the biosynthesis of the peptide anti-biotics gramicidin S, tyrocidine, and bacitracin,respectively, are repressed during most of theexponential growth phases of the producing ba-cilli. Biosynthesis of gramicidin S, a cyclic de-capeptide antibiotic, is carried out by two en-zyme fractions, the so-called heavy (280,000-dal-ton) and light (100,000-dalton) synthetases.Gramicidin S production occurs at the end ofthe exponential growth phase. The two enzymefractions form just before this time (108, 141,182). After a few hours, the enzyme activitiesdisappear.Tyrocidine synthetase is made of three com-

plementary enzymes, one light (100,000 daltons),one intermediate (230,000 daltons), and oneheavy (440,000 daltons). Formation of the tyro-cidine synthetase complex is repressed duringthe logarithmic growth phase and appears there-after (52).

In the cases of candicidin and bacitracin, thereis some evidence that repression is exerted atthe level oftranscription. Candicidin is a polyenemacrolide antibiotic produced by S. griseus; it issynthesized from acetate and propionate precur-sors in the form of activated malonyl-coenzymeA and methylmalonyl-coenzyme A units by theaction of a complex enzyme system called can-dicidin synthetase. Candicidin synthetase is sim-ilar in many respects to fatty acid synthetase(22, 167). In the candicidin fermentation, anti-biotic production starts after 18 h. If at any timecells are removed from the fermentation andplaced into a phosphate-free resting cell me-dium, they produce candicidin. However, if thesuspension medium contains an inhibitor oftranscription, such as rifampin, or an inhibitorof translation, such a chloramphenicol, candici-din formation depends on the age of the cultureused (113). When the culture is up to 10 h old,no candicidin is made; when it is 16 h old orolder, candicidin is made. These data indicatethat transcription of the candicidin synthetasegene(s) does not occur during the first 10 h ofgrowth.

Bacitracin is a peptide antibiotic produced byBacillus licheniformis. Purified bacitracin syn-thetase contains three complementary fractionswith molecular weights of 200,000, 210,000, and380,000 (49, 51, 83). In certain media, bacitracinformation by B. licheniformis occurs in the idi-ophase, after the exponential growth phase iscompleted. Production of bacitracin after maxi-mum growth requires the addition of high levelsofmanganese. The addition of inhibitors of tran-scription or translation (actinomycin D or chlor-amphenicol, respectively) with the manganese

or within 1 h thereafter completely inhibits bac-itracin formation. This result is to be expectedif bacitracin synthetase is made after growth butbefore the onset of antibiotic synthesis (197).Since bacitracin formation can be blocked byactinomycin D, an inhibitor of deoxyribonucleicacid-dependent ribonucleic acid polymerase, for-mation of bacitracin synthetase during growth isprobably blocked at the transcriptional level.

Unfortunately, nothing is known about thelongevity of messenger ribonucleic acid mole-cules coding for the enzymes which catalyzeantibiotic synthesis.

Inhibition of Antibiotic SynthetasesLate occurrence of antibiotic production could

also be due to inhibition of synthetase action.For example, in Cephalosporium acremonium,/?-lactam antibiotics (penicillin N and cephalo-sporin C) are made predominantly after growthhas been completed (93). However, resting cellstudies (done in the presence of cycloheximideto prevent protein synthesis by the resting cells)have shown that trophophase mycelia alreadycontain a very high level of fl-lactam-formingenzymes, even though antibiotic production bythese mycelia was miniimal in the fermentationat the time of harvest (29).Not all of the regulatory mechanisms control-

ling antibiotic biosynthesis are exerted at a sin-gle level (repression or inhibition). There is atleast one example in which both levels are in-volved in the biosynthesis of a single antibiotic.Thus, phosphate control of candicidin biosyn-thesis (see below) is exerted at two levels: (i) theonset of candicidin biosynthesis is repressed un-til phosphate is depleted from the broth, and (ii)candicidin synthesis is inhibited by phosphateafter the synthetase has been formed.

Inhibition in a Branched PathwayLeading to a Primary Metabolite and an

AntibioticThis subject has been reviewed recently (38,

129) and is considered here only briefly. Thebiosynthetic pathways of some antibiotics arebranched. They have an early common part,which then branches to the synthesis of a pri-mary metabolite on the one hand and to a sec-ondary metabolite on the other. In some cases,primary end product feedback inhibits the com-mon part of the pathway and thus inhibits an-tibiotic biosynthesis. Lysine inhibits penicillinproduction by Penicillium chrysogenum (61,139) and cephalosporin production by Paecilo-myces persicinus P-10 (23). When ["4C]lysine isused in the low-producing P. chrysogenumstrain Wis. 54-1255, more than 90% of the lysine

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in the broth must be exhausted before the onsetof penicillin biosynthesis (117). The molecularmechanism of the lysine regulatory effect hasbeen elucidated partially in recent years. It isnow well established that the penicillin biosyn-thetic pathway branches from the lysine path-way at the a-aminoadipic (or adenyl-a-aminoa-dipic acid) stage. Lysine inhibits in vivo biosyn-thesis of penicillin (139) and Demain and Ma-surekar (30) suggested that lysine acts at thelevel of homocitrate synthase, the first enzymein the lysine biosynthetic pathway. This sugges-tion has been confirmed by reversal studies (48)and by in vitro studies in which cell-free extractsof low- and high-producing P. chrysogenumstrains have been used (117).Another example involves the regulation of

candicidin biosynthesis by aromatic amino acids,especially tryptophan (115, 134). The inhibitionis cumulative; a mixture of phenylalanine, tyro-sine, and tryptophan inhibits candicidin biosyn-thesis by 74%, whereas the individual aminoacids inhibit by 28, 10, and 50%, respectively.This effect is exerted even in cells in whichprotein synthesis is blocked with chloramphen-icol. This inhibition is exerted at the p-amino-benzoate synthetase level.

INTRACELLULAR EFFECTORS ANDMECHANISMS THAT CONTROL THEONSET OF ANTIBIOTIC SYNTHESISSeveral mechanisms appear to be involved in

controlling the initiation of antibiotic biosyn-thesis. In one model, a small molecule acts as acorepressor or an inhibitor, repressing formationor inhibiting action of antibiotic synthetases.Therefore, the corepressor or inhibitor must bedepleted before antibiotic synthesis can occur.This model fits some of the experimental dataon carbon catabolite regulation, nitrogen metab-olite regulation, and phosphate control. In asecond regulatory model, an inducer or activatormust be synthesized by the producing culture oradded to it in order to initiate biosynthesis.

Carbon Catabolite RegulationGlucose, usually an excellent carbon source

for growth, interferes with the biosynthesis ofmany antibiotics (Table 1). During studies onfermentation medium development, polysaccha-rides or oligosaccharides are often found to bebetter than glucose as carbon sources for anti-biotic production (178). In a medium containingglucose plus a more slowly utilized carbonsource, glucose usually is used first in the ab-sence of antibiotic production. After glucose isdepleted, the second carbon source is then usedfor antibiotic biosynthesis (6, 24, 54).

Depending on the microorganism, the carbonregulatory effect may be exerted by rapidly usedcarbon sources other than glucose. For example,citrate is favored over glucose by the novobiocinproducer S. niveus, resulting in diauxic growth.Novobiocin production is suppressed during thephase of citrate utilization, but not during thesecondary phase of glucose utilization (104).Thus, we should not think of this phenomenonas a "glucose effect."The molecular mechanism of carbon catabo-

lite regulation may be related to growth ratecontrol of antibiotic biosynthesis (18, 133). Thus,slow addition of glucose to the fermentation toobtain a slow growth rate eliminates glucoseinterference with penicillin biosynthesis (179).Similarly, slow feeding of glucose to the fermen-tation stimulates formation of the polyene mac-rolide antibiotics candidin and candihexin (136).

In a few cases, glucose appears to repress aknown enzyme of an antibiotic biosyntheticpathway. Diminution of actinomycin biosyn-thesis by glucose is due to the repression ofphenoxazinone synthase, which is involved inthe formation of the phenoxazinone ring of ac-tinomycin (54). The mechanism behind the abil-ity of glucose to interfere with neomycin accu-mulation in Streptomyces fradiae is less obvious.

TABLE 1. Carbon catabolite regulation of antibioticbiosynthesis

InterferingAntibiotic carbon

source

Penicillin GlucoseActinomycin GlucoseStreptomycin" Glucose

SiomycinIndolmycinBacitracinCephalosporinC

Chlorampheni-col

ViolaceinProdigiosinMitomycinNeomycinKanamycinEnniatinPuromycinNovobiocinCandidin

Candihexin

ButirosinCephamycin

GlucoseGlucoseGlucoseGlucose

Glucose

GlucoseGlucoseGlucoseGlucoseGlucoseGlucoseGlucoseCitrateGlucose

Glucose

GlucoseGlycerol

Noninterferingcarbon

source(s)

LactoseGalactoseMannanSlowly fed

glucoseMaltoseFructoseCitrateSucrose

Glycerol

MaltoseGalactoseLow glucoseMaltoseGalactoseLactoseGlycerolGlucoseSlowly fed

glucoseSlowly fed

glucoseGlycerolAsparagine,

starch

Refer-ence(s)

178542882

977966, 6724

175

3316498121106

170104136

136

782

" Regulation refers to the enzyme mannosidostreptomyci-nase.

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Glucose appears to repress a phosphatase(s) re-quired for neomycin biosynthesis, but it alsostimulates inactivation of the antibiotic (121,122). O-demethylpuromycin methyltransferase,the last enzyme in the puromycin biosyntheticpathway, is repressed by glucose but not byglycerol (160, 169). Glucose also represses thebiosynthesis of kanamycin, an aminocyclitol an-tibiotic produced by Streptomyces kanamyceti-cus (10, 172). This effect is due to repression ofN-acetylkanamycin amidohydrolase, apparentlythe last enzyme in the kanamycin biosyntheticpathway (172).Recent studies on glucose regulation of peni-

cillin biosynthesis in P. chrysogenum indicatethat glucose represses (but does not inhibit)incorporation of ['4C]valine into penicillin (G.Revilla and J. F. Martin, unpublished data).

In streptomycin biosynthesis, glucose inter-feres with the interconversion of the less activecompound mannosidostreptomycin into themore active compound streptomycin by repress-ing mannosidostreptomycinase (82). Similarly,glucose represses cephalosporin C acetylhydro-lase, which converts cephalosporin C into theless active compound deacetylcephalosporin C(69).Although some older reports suggest that the

biosyntheses of neomycin, siomycin, and peni-cillin G are subject to carbon catabolite inhibi-tion rather than repression (25, 97, 142), theexperimental evidence does not unequivocallyprove these claims, which are based on inhibi-tion of antibiotic biosynthesis after glucose ad-dition to cells committed to antibiotic produc-tion. Since the experiments were long, the inter-ference with antibiotic synthesis could havebeen due to repression of labile antibiotic syn-thetases which must be replenished continu-ously. However, data from recent short-termexperiments on production of penicillin N byresting cells of C. acremonium strongly suggestsome type of catabolite inhibition by glucose(29). This phenomenon appears to be complexand may involve ammonia regulation as well.Mechanisms other than catabolite regulation,

such as decreased pH or depletion of dissolvedoxygen, could be involved in interference of an-tibiotic biosynthesis by carbon sources. Glucoseinterference in bacitracin production has beenreported to be due to decreased pH (66, 67).However, the interference is not due to pH perse, but to the undissociated forms of acetic andpyruvic acids, which accumulate when culturesare grown with a high glucose concentration.Carbon catabolite repression of inducible cat-

abolic enzymes in some microorganisms involvescyclic adenosine 3,5-monophosphate (cAMP) as

a positive effector (154). A high glucose concen-tration indirectly inhibits the activity of adenylcyclase, thereby decreasing the intracellularlevel of cAMP. cAMP, a positive effector, inter-acts with a cAMP receptor protein; the cAMPreceptor protein-cAMP complex binds to thepromotor sites of operons coding for inducibleenzymes and thus activates gene transcription.

It is not known whether cAMP is involved ina single case of carbon catabolite regulation ofantibiotic synthesis. cAMP does not reverse glu-cose repression of penicillin biosynthesis in P.chrysogenum (Revilla anTd Martin, unpublisheddata). On the other hand, it is claimed thatcAMP relieves glucose repression of N-acetyl-kanamycin amidohydrolase in S. kanamyceti-cus. cAMP and its binding protein (and cyclicguanosine 3,5-monophosphate) have been foundin S. hygroscopicus, the producer of turimycin(a 16-member macrolide antibiotic), but it is notknown whether these compounds play any rolein regulating turimycin biosynthesis (55). cAMPcontent drops at the onset of turimycin forma-tion, and added cAMP stimulates growth andinterferes with turimycin synthesis. This inter-ference could be caused by phosphate regula-tion. The cAMP content in S. griseus drops to10% of its trophophase peak level before strep-tomycin formation (163). These observationssuggest that a high cAMP level does not relieverepression of antibiotic formation, in contrast tothe inducible enzymes in enteric bacteria. In-stead, antibiotic synthetases may be turned offby high cAMP levels; this action may be con-nected more closely to phosphate regulationthan to carbon regulation.

Nitrogen Metabolite RegulationRecently, a regulatory mechanism that con-

trols the use of nitrogen sources has been re-ported in bacteria, yeast, and molds (39, 119).Ammonia (or some other readily used nitrogensource) represses enzymes involved in the use ofother nitrogen sources; these enzymes includenitrite reductase, nitrate reductase, nicotin-amide adenine dinucleotide-dependent gluta-mate dehydrogenase, arginase, ornithine trans-aminase, extracellular protease, acetamidase,threonine dehydratase, allantoinase, and thoseenzymes dealing with purine degradation, trans-port of urea and glutamate, and histidine use.

In enterobacteria, nitrogen metabolite regu-lation appears to involve glutamine synthetase(EC 6.3.1.2). This enzyme not only functions inglutamine biosynthesis, but also regulates thesynthesis of enzymes involved in assimilatingnitrogen compounds (119). Nitrogen metaboliterepression in Klebsiella aerogenes is mediated

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through the intracellular levels ofglutamine syn-thetase.

In fungi, an enzyme protein also appears to beinvolved in nitrogen metabolite regulation, butin an opposite way. Whereas glutamine synthe-tase in enteric bacteria is required to derepress,nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase in fungi ap-pears to be necessary to repress enzymes ofnitrogen metabolism. In molds and yeasts, nitro-gen metabolite repression requires NH4', a-ke-toglutarate, and a catalytically active glutamatedehydrogenase (nicotinamide adenine dinucleo-tide phosphate specific) (39). Mutants lackingthis enzyme are derepressed.Few studies have dealt specifically with nitro-

gen metabolite regulation of antibiotic biosyn-thesis, but several reports in the literature indi-cate that antibiotic biosynthesis may be sug-gested by ammonia and other rapidly utilizednitrogen sources. Erythromycin production pro-ceeds as long as there is carbon source in thefermentation, but if a nitrogen source (ammo-nium chloride, glycine, or soybean meal) isadded, erythromycin production is depressed(176). The addition of soybean meal extract tocandicidin or candihexin fermentations duringthe idiophase interferes with antibiotic synthe-sis, but increases respiration (136).

Screening of nitrogen sources for antibioticproduction in chemically defined media has fre-quently resulted in the selection of slowly me-tabolized amino acids, the use ofwhich probablyresults in nitrogen limitation. A classic case isthe superiority of proline in the streptomycinfermentation (40).Two recent publications describe nitrogen me-

tabolism of Streptomyces noursei in relation tothe biosynthesis of nourseothricin, a strepto-thricin-type antibiotic (63, 65). Glutamine syn-thetase in this microorganism is repressed by ahigh concentration of ammonium ions. Afterammonium ion is depleted from the medium,derepression of glutamine synthetase occurs. Al-though no connection between glutamine syn-thetase and nourseothricin biosynthesis hasbeen established, o-aminobenzoic (anthranilic)acid stimulates nourseothricin biosynthesis andhas a marked effect on the regulation of enzymesof nitrogen metabolism. The intracellular levelof ammonium ions in S. noursei is influenced bythe action of alanine dehydrogenase. Ammo-nium liberated by alanine oxidation is availablefor biosynthesis of glutamine via glutamine syn-thetase. o-Aminobenzoic acid represses both glu-tamine synthetase and alanine dehydrogenasewhile stimulating formation of glutamate dehy-drogenase and the antibiotic (65).

The activity of the nicotinamide adenine di-nucleotide-specific alanine dehydrogenase of S.hygroscopicus, the producer of the macrolideantibiotic JA 6599, also appears to be correlatedinversely with antibiotic production (64).

Cephalosporin biosynthesis in streptomycetesis subject to nitrogen metabolite regulation (3,57); the control mechanism may involve gluta-mine synthetase and alanine dehydrogenase (1).

Phosphate RegulationPhosphate is the crucial growth-limiting nu-

trient in many antibiotic fermentations; it isvirtually exhausted during growth of S. griseus(a candicidin producer) 2 h before the onset ofcandicidin synthesis (113). Phosphate is also de-pleted during growth of S. aureofaciens beforethe onset of tetracycline production. Extracel-lular phosphate remains at very low levels dur-ing the entire idiophase of candicidin fernenta-tions. If 10 mM phosphate is added at the startof a candicidin fermentation, extracellular phos-phate is not depleted; growth continues through-out the fermentation, and no antibiotic synthesisoccurs. With increasing concentrations of addedphosphate, phosphate depletion is delayed, as isthe onset of antibiotic synthesis (Fig. 2). Thus,candicidin synthesis is initiated by phosphatedepletion. A similar phenomenon occurs in van-comycin fermentations (143). Phosphate also in-hibits candicidin synthesis once it has started(116, 130).Phosphate regulates the syntheses of antibiot-

ics belonging to different biosynthetic groups;these include peptide antibiotics, polyene mac-rolides, tetracyclines, and biosynthetically com-plex antibiotics. Industrial production of theseantibiotics is carried out at growth-limiting con-

200

E 150

Z 1000

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0

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FIG. 2. Effect of increasing phosphate concentra-tion (added at zero time) on candicidin formation byS. griseus. Values labeling lines represent the concen-trations (millimolar) of phosphate added. Redrawnfrom reference 113.

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centrations of inorganic phosphate. Phosphatein concentrations ranging from 0.3 to 300 mMgenerally supports extensive cell growth, butconcentrations of 10 mM and above suppressthe biosynthesis of many antibiotics (128). Be-cause the various antibiotics regulated by phos-phate are synthesized via different pathways,many different mechanisms may exist, or else acommon regulatory effector may act on the var-ious biosynthetic pathways (127). Phosphate ad-dition not only interferes with antibiotic synthe-sis but also after several hours causes a reversalof nongrowing, antibiotic-producing cells backto a growing, nonproducing state (116).

In the last few years, studies have been carriedout on the regulatory effect of phosphate oncandicidin biosynthesis by S. griseus (128, 130).An important question is whether intracellularorthophosphate is the ultimate effector orwhether it merely regulates the level of someother intracellular effector that controls expres-sion of antibiotic biosynthesis.The intracellular adenosine triphosphate

(ATP) level of S. griseus increases rapidly afterthe addition of 10 mM phosphate to antibiotic-producing cells, doubling within 5 min of phos-phate addition. This increase occurs before theinhibition of antibiotic synthesis, which is de-tected 15 min after phosphate addition. The rateof protein or ribonucleic acid synthesis does notchange during this time (130). This finding sug-gests that ATP may be the intracellular effectorthat controls antibiotic synthesis. The possibleinvolvement of ATP in controlling antibioticbiosynthesis is also supported by the data ofSilaeva et al. (174) and Janglova et al. (85), whoreported that ATP levels are lower in improvedantibiotic-producing strains than in their low-producing ancestral strains. Fynn and Davison(53) also point out the possible involvement ofATP in regulating antibiotic biosynthesis. Theinhibition of candicidin biosynthesis in S. gri-seus by the exogenous ribonucleotides (includingcyclic nucleotides) of adenine, guanine, cytosine,and uracil, but not by ribonucleosides or theirbases, has been described previously (131). Up-take studies indicate that the nucleotides arecleaved during uptake and that their effects aredue to the released phosphate (132).Whether ATP concentration or adenylate en-

ergy charge in cells is the true regulatory param-eter of enzymes is still controversial. Energycharge, as defined by Atkinson and Walton (5),is one-half the number of anhydride-boundphosphates per adenosine moiety, i.e.:

ATP + 0.5 adenosine diphosphate (ADP)ATP + ADP + AMP

it is a linear measure of the amount of energystored at any time in the adenylate system. Itsnormal values during exponential growth ofEscherichia coli (21), Bacillus subtilis (81), andSaccharomyces cerevisiae (7) are 0.8, 0.7, and0.8 to 0.9, respectively. Atkinson and co-workersmaintain that the energy charge is a regulatoryparameter coordinating energy-utilizing and en-ergy-generating metabolic pathways and is moreimportant than the absolute concentration ofATP, but this view is not universally accepted(162).The energy charge of antibiotic-producing

resting cells of S. griseus is about 0.8 (135). Afterthe addition of 10 mM phosphate to the system,the energy charge increases only slightly, toabout 0.85. On the other hand, the intracellularATP concentration doubles or triples. The pos-sibility exists, but certainly is not proven, that ahigh ATP level inhibits candicidin synthesis.This postulated role of ATP is consistent withdata from time course studies on ATP concen-tration during antibiotic fermentations in whicha rapid decrease in intracellular ATP occursbefore the onset of antibiotic synthesis (113).Phosphatase regulation also appears to be in-

volved in antibiotic biosynthesis. In the biosyn-thetic pathways of certain antibiotics, such asstreptomycin (146), viomycin (152), and neo-mycin (120), some intermediates are phospho-rylated, whereas the end products are not. Ingeneral, microbial phosphatases that cleavephosphorylated intermediates are often regu-lated via feedback inhibition or repression byinorganic phosphate. Streptomycin biosyn-thesis, which is markedly inhibited by phos-phate, includes at least three phosphate-cleavingsteps in the formation of the streptidine moiety(28). Miller and Walker (145) reported that aphosphorylated derivative of streptomycin ac-cumulates in cultures of S. griseus growing inexcess inorganic phosphate. The phosphorylatedderivative, which contains a phosphate ester atcarbon 6 of the streptidine moiety, is biologicallyinactive (146, 148, 149). Experiments of Walkerand co-workers suggest that the phosphategroup is introduced during biosynthesis of thestreptidine moiety (192). The dihydrostreptosemoiety is transferred enzymatically from deoxy-thymidine diphosphate-dihydrostreptose tostreptidine 6-phosphate to form O-a-L-dihydro-streptose (1-4)-streptidine 6-phosphate (103).Streptomycin phosphate is probably an obliga-tory intermediate in streptomycin biosynthesis(148). Streptomycin phosphatase, an idiophaseenzyme present only in streptomycin producers,is inhibited, but not repressed, by phosphate(194).

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A similar involvement of phosphatase controlhas been described in the biosynthesis of neo-mycin B by S. fradiae (120). The alkaline phos-phatase of S. fradiae is inhibited and repressedby inorganic phosphate (121). This enzyme issynthesized late in the fermentation, and thereappears to be a direct relationship between en-zyme activity and neomycin formation. The ap-pearance of alkaline phosphatase is due to denovo protein synthesis, which was demonstratedby the inhibition of its synthesis with chloram-phenicol (8).The alkaline phosphatase of Proactinomyces

fructiferi var. ristomicini, the organism whichproduces the antibiotic ristomycin, is completelyrepressed at concentrations of inorganic phos-phate that inhibit antibiotic production (183).Gersch et al. (56) have claimed that cAMP

reverses phosphate inhibition of secondary me-tabolism in S. hygroscopicus. Such is not thecase with candicidin biosynthesis in S. griseus;phosphate inhibition of candicidin formation ineither resting or growing cells is increased fur-ther by cAMP (G. Naharro and J. F. Martin,unpublished data).

InductionEnzyme induction is a well-known regulatory

mechanism in primary metabolism. Inductionappears to be involved in controlling the biosyn-thesis of some antibiotics and other secondarymetabolites, although the phenomenon has notbeen studied extensively.

It is sometimes difficult to determine whetherthe stimulatory phenomenon is a true inductioneffect or merely a precursor effect. We includeas inducers those compounds which stimulateantibiotic biosynthesis when added during thegrowth phase before the idiophase, but not whenadded to idiophase cells in which protein syn-thesis has been inhibited by the addition of aninhibitor. Furthermore, these compounds shouldbe replaceable by a nonprecursor analog. On theother hand, precursors stimulate antibiotic syn-thesis when added during the idiophase, evenwhen protein synthesis has been blocked.An intriguing induction-like phenomenon is

the stimulation of streptomycin biosynthesis byA-factor (2-S-isocapryloyl-3-R-hydroxymethyl-y-butyrolactone) (Fig. 3) (99). It has been re-ported that 1 ,ig of pure A-factor added at thetime of inoculation to a mutant of S. griseusblocked in streptomycin biosynthesis inducesthe production of 1 g of streptomycin; i.e., theinduction coefficient is 106 (96). The A-factor isformed by all wild-type and industrial strains ofS. griseus which have been investigated. Mu-tants unable to synthesize the inducer lack the

ability to form streptomycin. A-factor is alsoproduced by Streptomyces bikiniensis, but notby other species that synthesize streptomycin,such as S. galbus and Streptomyces mashuensis(94, 96). The stimulatory effect of A-factor isintense when this compound is added at thetime of inoculation, but it is absent when thecompound is added after 48 h. A short treatmentperiod of 3 to 4 min with A-factor, followed bywashing, is sufficient to induce streptomycin for-mation. A-factor restored the ability to producestreptomycin to 114 of 119 nonproducing mutantstrains examined. Such mutants are not capableof synthesizing the streptidine moiety of strep-tomycin unless supplemented with A-factor. A-factor is also involved in S. griseus differentia-tion. Antibiotic-producing strains differentiatenormally, releasing aerial spores in solid mediaor in submerged cultures. Mutants are not ableto differentiate unless supplemented with A-fac-tor. The addition of A-factor to the mutants alsocauses formation of well-developed intracellularmembranes and tubular structures which aresimilar to those in the producing strains.The molecular mechanism of induction of

streptomycin biosynthesis by A-factor is notknown. However, there are some data that maypartially explain its effect on enzymes of carbo-hydrate metabolism. Thus, the activity of glu-

FRUCTOSE-6-(

IGLUCOSAMINE - 6-®

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GLUCOSE

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GLUCOSE-6-(Df) 6-- GLUCONATE

PENTOSE -®PATHWAY OF

GLUCOSE- I- INOSITOL- I- PRIMARYMETABOLISM

TDP-GLUCOSE

STREPTOSE

ISTREPT IDINE

FIG. 3. Mechanisms ofA-factor action asproposedby Khokhlov and Tovarova (95). TDP, Thymidinediphosphate.

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cose-6-phosphate:nicotinamide adenine dinucle-otide phosphate-oxidoreductase (glucose-6-phosphate dehydrogenase) (EC 1.1.1.49) is re-ported to be very high in nonproducing mutants,whereas it is practically undetectable in high-producing strains (96). The addition of A-factorto nonproducing strains immediately decreasesglucose-6-phosphate dehydrogenase activity;such an addition, however, does not affect thepure isolated enzyme. This result suggests thatA-factor induces the formation of another com-pound, which in turn inhibits glucose-6-phos-phate dehydrogenase. Khokhlov and Tovarova(96) suggest that the compound might be aden-osine diphosphoribose, a breakdown product ofnicotinamide adenine dinucleotide phosphatevia nicotinamide adenine dinucleotide phospha-tase action. They further propose that when thepentose phosphate pathway is blocked at theglucose-6-phosphate dehydrogenase stage, glu-cose is preferentially transformed into the strep-tomycin moieties (streptose, streptidine, and N-methyl-L-glucosamine) instead of undergoingoxidation (Fig. 3).Another induction-like phenomenon is the

stimulation of cephalosporin C biosynthesis bymethionine in C. acremonium. Methionine ap-pears to be both a precursor and an inducer ofcephalosporin biosynthesis (35). Norleucine, thenonsulfur analog of methionine (31), is also aninducer. Results of Drew and Demain (35-37)with sulfur amino acid auxotrophs of C. acre-monium supported the importance of methio-nine as a regulatory effector of cephalosporinbiosynthesis and emphasized the importance ofendogenous methionine in fermentations con-ducted without exogenous methionine. Figure 4shows the pathway of sulfur utilization in fungi.In a double mutant (strain 11-8) of C. acremon-ium blocked in the paths from S042- to cysteineand from cysteine to methionine, norleucine wasable to replace excess methionine in stimulatingcephalosporin production. The stimulatory ac-tivity of norleucine is critically significant sincethis compound contains no sulfur.The recent data of Treichler et al. (184) agree

with the results of Drew and Demain (35, 36).Thus, a mutant (OAH-/SeMer) that was defec-tive in step 8 (Fig. 4) and thus grew on methio-nine, homocysteine, and cystathionine, but noton cysteine or inorganic sulfur sources, was notstimulated in antibiotic production by excesscysteine or sulfate in a medium containing a lowbut growth-sufficient concentration of methio-nine. Another mutant (SMcyj/MeSer), whichwas deficient in step 9 (Fig. 4), was depressed incephalosporin synthesis from sulfate but notfrom methionine. Also, a mutant (OAS-)

CEPHALOSPORIN C

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FIG. 4. Probable pathway ofsulfur in D. acremon-ium. SAM, S-adenosylmethionine; SAH, S-adenosyl-homocysteine.

blocked in step 7 showed an increased ability toproduce cephalosporin from excess sulfate; thiswas presumably due to diversion of sulfur tomethionine via steps 9 and 12 when step 7 wasgenetically blocked.

Despite the general agreement between thedata of Drew and Demain (35, 36) and those ofTreichler et al. (184), the two groups have ten-tatively reached somewhat different conclusions.Drew and Demain favor the hypothesis thatmethionine acts as an inducer of one or moref8-lactam synthetases; Treichler et al. (184) be-lieve that cystathionine-y-lyase (Fig. 4, step 17)is crucial in the methionine effect, this cysta-thionine-cleaving enzyme somehow transferringthe cysteine moiety into the cephalosporin bio-synthetic pathway (as "active cysteine"?). Bothgroups have data on mutants to support theirpositions. Drew and Demain (37) used a doublesulfur auxotroph (strain H) thought to beblocked both between steps 2 and 8 and in step17; it grows on cysteine but not on methionine,homocysteine, cystathionine, or sulfate. Sincesuch a mutant should not be able to convertcystathionine to cysteine and presumably lackscystathionine-y-lyase, the key enzyme of thehypothesis of Treichler et al. (184), its antibioticproduction should not be stimulated by methi-onine sources unless methionine induces cepha-losporin-forming enzymes. Mutant H was indeedstimulated by methionine dipeptides (which hadto be used instead of methionine for technical

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reasons involving permeation), thus supportingthe role of methionine as inducer. On the otherhand, mutant 8650/113 of Treichler et al. (184),which lacks cystathionine--y-lyase (step 17) andgrows on cysteine or inorganic sulfur sources(but not on methionine, homocysteine, or cys-tthionine), failed to respond with antibiotic pro-duction to methionine added to a sulfate-con-taining medium. Also, when sulfate was the solesulfur source, mutant 8650/113 produced muchless cephalosporin than its wild-type parent,again indicating the importance of cystathio-nine-y-lyase. Further studies will be necessaryto settle this controversy.Methionine induces fragmentation of the my-

celium into arthrospores simultaneously withinduction of cephalosporin biosynthesis. It is notclear, however, whether the antibiotic is an ef-fector required for morphological differentiationor whether cephalosporin production is merelya consequence of morphological differentiation.The stimulatory effect of 5,5-diethylbarbitur-

ate (barbital) on the biosynthesis of rifamycin(111) and anthracycline antibiotics (107) mayalso be an induction effect. Barbiturates, espe-cially diethylbarbiturate, direct rifamycin bio-synthesis toward the formation of rifamycin B.Barbital is not a precursor of rifamycins sincebarbital labeled either in the ring or in the sidechain is neither incorporated into rifamycin normetabolized to malonate by Nocardia mediter-ranei (102, 111). More than 90% of the labeledbarbital can be recovered at the end of thefermentation as undegraded barbital. The re-maining small percentage is in the form of fourtransformation products, none of which exhibitsstimulatory activity. Barbital only exerts thiseffect when added very early in the fernenta-tion; barbital added after 48 h of fermentation isineffective (125). Studies on the effect of barbi-tal, in which washed mycelium of N. mediter-ranei was used, led to the conclusion that bar.bital added during the growth phase increasesthe activity of the enzymatic system transform-ing rifamycin SV into rifamycin B (111).

Production of the anthracycline galirubin byStreptomyces galileus is also stimulated by bar-bital. The stimulation is higher (up to threefold)in low-producing strains than in high-producingmutants. Stimulation occurs only if barbital isadded before 36 h of cultivation, and the effectis more intense under conditions of limited aer-ation. Barbital may affect specific enzymes thathydroxylate aklavinone (107); its regulatory ef-fect is not a general one, since barbital does notstimulate production of daunomycinone glyco-sides by Streptomyces coeruleorubidus (14).Compounds that stimulate tetracycline bio-

synthesis include benzylthiocyanate, a-naph-thylacetic acid, fi-indoleacetic acid, phenylaceticacid, and p-chlorophenoxyacetic acid (186). Thestimulatory effect ofbenzylthiocyanate has beenstudied in some detail (75-77). At a benzylthio-cyanate concentration of 10 pm, the enhance-ment of tetracycline production is accompaniedby decreases in sugar utilization and glucoseoxidation through the glycolytic pathway (77).Benzylthiocyanate also decreases the formationof the enzymes of the tricarboxylic acid cycle; itsstimulatory effect is stronger in low-producingthan in high-producing strains.

Benzylthiocyanate stimulates tetracyclinebiosynthesis only if added to the culture duringthe first 12 h offermentation, when the tetracenenucleus has not yet been formed. This findingsuggests a regulatory effect on primary metab-olism and/or an induction of tetracycline-syn-thesizing enzymes. Benzylthiocyanate also stim-ulates chlortetracycline production in restingcells of S. aureofaciens (34).Other possible examples of induction are the

increase in staphylomycin (synonym, virginia-mycin) biosynthesis by an inducer produced byStreptomyces virginiae (198, 199) and the stim-ulation of fosfomycin biosynthesis by methio-nine (166). The staphylomycin biosynthesis in-ducer is formed just before the appearance ofthe antibiotic. Surprisingly, most of this inducersubstance is excreted into the broth. It wassuggested that the quantity of inducer formedwas much larger than required for induction ofstaphylomycin production. The level of inducerand therefore the production of staphylomycinare greatly inhibited by the previous addition ofstaphylomycin to the culture. This result sug-gests that staphylomycin represses its own bio-synthesis by inhibiting fornation of the staphy-lomycin inducer (198, 199).

CESSATION OF ANTIBIOTICBIOSYNTHESIS

The duration of antibiotic production differsin genetically distinct producing strains and un-der different environmental conditions. Afterformation of the antibiotic synthetases, the rateof antibiotic biosynthesis is linear for a period oftime. In some antibiotic-producing cultures, thephase of active antibiotic synthesis is rathershort, lasting 4 to 20 h. In the actinomycetes andmolds that produce antibiotics, the productionphase is usually much longer than the tropho-phase. The production phase may be prolongedfor several days by continuously or intermit-tently providing nonrepressive or noninhibitorylevels of a carbon source. For example, the can-didin and candicidin fermentation may be main-

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tained for 300 h in phosphate-limited culturesby slow feeding of glucose (116, 136). Industrialproduction of penicillin is carried out for morethan 200 h, and fedbatch systems have beenintroduced recently in which penicillin is syn-thesized for at least 10 days (157). Even in thesecases, however, the rate of antibiotic formationdecreases with time.

Cessation of antibiotic synthesis may not bedue to loss of viability of the producing cells.Lysis of antibiotic-producing cells is avoided aslong as the carbon source is not depleted. Thereare at least three possible reasons for the cessa-tion of antibiotic biosynthesis: (i) irreversibledecay of one or more enzymes of the antibiotic-synthesizing pathway; (ii) the feedback effect ofthe accumulated antibiotic; and (iii) depletion ofintermediary precursors of the antibiotic. Exper-imental evidence supports the first two possibil-ities.

Irreversible Decay of AntibioticSynthetases

The activities of the synthetases of peptideantibiotics decrease rapidly a few hours after theonset of antibiotic production. In vivo stabiliza-tion of such enzymes would allow for a greatlyprolonged period of product formation. Tyroci-dine synthetase activity disappears from the sol-uble portion of Bacillus brevis cells and appearsto associate with the forespore membrane atstage IV of the sporulation process (112). Themembrane-bound synthetase activity associatedwith the forespore disappears a few hours later.Similarly, soluble bacitracin synthetase occursonly in a short period during the growth of B.licheniformis. The soluble bacitracin synthetasebecomes predominantly cell bound later in thegrowth phase (50). This may represent a naturalimmobilization of an enzyme complex, whichprotects the complex against intracellular pro-teolytic activities appearing at the end of thegrowth phase (173).The molecular mechanism of inactivation of

the antibiotic synthetases is unknown. In vitroincubation of the heavy gramicidin S synthetaseat 37°C for 1 h resulted in an 80% loss of gram-icidin S-synthesizing activity (46, 100). Klein-kauf and Koischwitz (101) reported that thisinactivation was due to the presence of a pro-tease in the crude extract; they also observedthat the heavy enzyme was divided into subunitsof different sizes by proteolytic enzymes andthat this could be prevented by adding a mixtureof protease inhibitors. However, Friebel and De-main (46) observed that the inactivation ofgramicidin S synthetase in frozen and thawedcells or in cell-free extracts was oxygen depend-

ent and independent of protease action. Inhibi-tors of protease and of energy metabolism hadno effect on the inactivation process, but inacti-vation was prevented when cells were shaken innitrogen or helium instead of air. The L-orni-thine- and D-phenylalanine-activating activitiesof the gramicidin S synthetase complex werealso lost during aeration of the cells. This resultdiffers from that of Kleinkauf and Koischwitz(101), who demonstrated a loss of antibiotic-forming activity but a retention of amino acid-activating activity. These discrepancies suggestthat there may be two different types of inacti-vation mechanisms for gramicidin S synthetase.The oxygen-dependent inactivation may involveoxidation and inactivation of some of the sulf-hydryl groups which exist in gramicidin S syn-thetase (110).

It is interesting to note that removal of oxygenin vivo by sparging with nitrogen prevents in-activation of gramicidin S synthetase (46, 47).Oxygen is required for growth, but it is possiblethat control of dissolved oxygen, to provide ahigh degree of oxygen transfer during exponen-tial growth followed by a moderate degree ofoxygen transfer during the stationary phase,could improve antibiotic production.

Feedback Regulation by Anitbioticsof Their Own Biosynthesis

The role of feedback regulation in controllingspecial metabolism has been obscured by a lackof knowledge of the enzymes involved in anti-biotic biosynthesis. Recently however, it hasbecome evident that several antibiotics inhibittheir own biosynthesis. Examples include chlor-amphenicol (87, 124), aurodox (114), cyclohexi-mide (105, 106), staphylomycin (198), ristomycin(42), puromycin (170), fungicidin (180), candi-hexin (136), and mycophenolic acid (147). Areport by Gordee and Day (59) on the inhibitionof penicillin biosynthesis by penicillin itself hasbeen confirmed recently by us (165).

In most cases, the molecular mechanism offeedback regulation is not known. In a few cases,however, antibiotics are known to inhibit theactivity of their own synthetases. End productregulation of mycophenolic acid biosynthesis inPenicillium stoloniferum involves regulation ofS-adenosylmethionine:demethylmycophenolicacid O-methyltransferase, the enzyme that cat-alyzes the final step in the synthesis of myco-phenolic acid. This activity of the enzyme isinhibited 68% by 25 mM mycophenolic acid(147). Similarly, puromycin inhibits the last en-zyme of its biosynthetic pathway, S-adenosyl-methionine:O-demethylpuromycin 0-methyl-transferase (170). The feedback mechanism is

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also known in the feedback control of arylaminesynthetase by chloramphenicol (87). Arylaminesynthetase is the first enzyme unique to thebiosynthesis of chloramphenicol; it convertschorismic acid (a common aromatic intermedi-ate) into p-aminophenylalanine (86). This en-zyme, which has been found only in chloram-phenicol-producing cultures, is fully repressedwhen the chloramphenicol concentration in themedium reaches 100 mg/liter. This concentra-tion of chloramphenicol affects neither cellgrowth nor the formation of other enzymes ofthe aromatic amino acid pathway, such as chor-ismate mutase, prephenate dehydratase, and an-thranilate synthetase.The synthesis-inhibiting level of antibiotic in

a particular producing strain is usually similarto the production level of that strain. Strepto-myces sp. 3022 produces 120 ,Lg of chloramphen-icol per ml, which is approximately the concen-tration that totally inhibits antibiotic synthesis.Gordee and Day (59) showed that 15 mg ofpenicillin per ml completely inhibits productionby the high-producing mutant E-15, whereas 2mg/ml is sufficient to inhibit penicillin produc-tion by strain Q176 (which produces 420 jg/ml)and 200 ,g/ml completely abolishes penicillinproduction by strain NRRL 1951 (which pro-duces 125 ,ug/ml). Using a resting cell systemthat measured the incorporation of [14C] valineinto penicillin in short-term experiments, Revillaet al. (165) found that exogenous penicillin in-hibits synthesis of penicillin when added at thetime of inoculation, but has less or no effectwhen added later. This change in the sensitivityto penicillin apparently is related to a decreasein the uptake ofexogenous penicillin by the cells.The concentration of aurodox required to blockfurther antibiotic synthesis completely is about400 ,ug/ml, the same concentration of aurodoxnormally produced by a culture of the producingorganism (114).An interesting exception is the suppression of

staphylomycin synthesis by small amounts (5Ag/ml) of the minor component staphylomycinS when added at the beginning ofthe incubation,even though the culture synthesizes as much as300 ,g/ml. Inhibition by exogenous staphylo-mycin is exerted during a sensitive stage in themiddle of the exponential phase. As mentionedabove, the inhibitory effect of staphylomycinmight be exerted at the level of formation of theinducer of antibiotic synthesis (198).

GENETIC MANIPULATION OF THEMECHANISMS CONTROLLINGANTIBIOTIC BIOSYNTHESIS

Strain development programs in industry

have markedly increased the production abilityof many antibiotic-producing cultures. Most ofthese efforts have been carried out by randomtesting of survivors of mutagenesis for produc-tion on agar plates or in liquid cultures (26).Examinations of such strains have revealed de-creases in regulation of antibiotic production.For example, valine (a precursor of penicillin)inhibits the activity of acetohydroxyacid synthe-tase, the first enzyme in the valine biosyntheticpathway (62). The regulatory effect is not ex-erted, however, by the other branched aminoacids (leucine and isoleucine) whose biosyn-thesis involves the same pathway. A high-pro-ducing mutant of P. chrysogenum, which wasdescribed by Goulden and Chattaway (62), wasfound to be less sensitive to valine inhibition ofacetohydroxyacid synthetase than its ancestralstrain.Most of the mutants obtained in these strain

development programs are probably regulatorymutants. The concept that control mechanismscan be bypassed by mutations is finally beingexplored in strain improvement programs. Al-though no one has yet derived a method to selectdirectly for high production of secondary metab-olites, attempts toward more rational screeningprocedures have become common (see below).Feedback regulation is probably decreased in

mutants ofPseudomonas aureofaciens resistantto tryptophan analogs; some are overproducersof pyrrolnitrin (43). The increased production ispresumably due to removal of the regulatorymechanism that controls tryptophan synthesis,since tryptophan is a precursor of pyrrolnitrin.A similar case is the trifluoroleucine-resistantmutant of Streptomyces lipmanii (58) that ex-cretes up to sixfold more cephamycin than itsparent strain. This mutant is probably deregu-lated in the branched amino acid biosyntheticpathway, which would supply valine for cepha-mycin production.

Suppression of an auxotrophic mutation by asecond mutation sometimes yields feedback-re-sistant mutants in primary metabolism. Thesuppression of a cys auxotrophic mutation in S.lipmanii led to improved production of cepha-mycin (58). Suppression of a met auxotrophicmutation in Streptomyces viridifaciens (41) andof an ilv mutation in S. antibioticus (161) im-proved production of chlortetracycline and ac-tinomycin, respectively.Another strategy of eliminating control mech-

anisms of antibiotic biosynthesis is to suppressantibiotic-nonproducing mutants back to pro-duction. Dulaney and Dulaney (41) reportedthat a significant proportion of such revertantsof S. viridifaciens are overproducers of chlortet-

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racycline. Using this strategy, Unowsky andHoppe (185) recently obtained a 20% increase inaurodox production.Although the above-described screening

methods yield superior producers, they are stillrather indirect methods. Very recently, therehas been an attempt to screen directly for amutant deregulated in a control mechanism ofmajor importance in the formation of a particu-lar antibiotic, i.e., phosphate control of candici-din production. Martin et al. (138) devised amethod to visualize antibiotic production onagar in the presence of phosphate. They ob-tained mutants of S. griseus that were altered inphosphate regulation so that phosphate nolonger inhibited candicidin biosynthesis (138).Some of these mutants are higher candicidinproducers than the wild type in both phosphate-supplemented and nonsupplemented productionmedia. These mutants are not phosphate perme-ability mutants but rather appear to be deregu-lated in phosphate control of candicidin synthe-sis. Phosphate-deregulated mutants may haveconsiderable industrial significance, permittingantibiotic fernentations to be done in complexmedia with high phosphate contents.Knowledge of the biochemical genetics of an-

tibiotic biosynthetic pathways has advancedslowly compared with the rapid progress in basicmolecular biology and genetics. However, devel-opment of fundamental biochemistry and ge-netics has provided a sound basis for rapid re-search progress in the genetics and molecularbiology of antibiotic biosynthesis. Of special in-terest will be the genetic engineering of anti-biotic-producing strains, including the manipu-lation of plasmids containing structural or reg-ulatory genes coding for antibiotic production(74).

SUMMARYUnlike primary (or general) metabolites,

which are produced or required by all microor-ganisms, secondary (specific) metabolites areproduced only by small groups of organisms andare not required for growth in culture, althoughthey probably have survival value in nature.Secondary metabolites are structurally diverseand unusual, generally are produced in mixtureswith other members of the same chemical fam-ily, and usually are formed at low specific growthrates.

In batch culture, some processes leading tothe production of antibiotics are sequential; i.e.,they exhibit a distinct growth phase (tropho-phase) followed by a production phase (idi-ophase). In other processes, trophophase andidiophase overlap. Timing depends on the nutri-

tional environment presented to the cultureand/or on the growth rate.

Antibiotic production in nature may allowproducing organisms to compete effectively withother forms of life or to differentiate (91). Delayin antibiotic production until after trophophaseis useful to a producing organism, since theorganism is sensitive during growth to its ownantibiotic. Resistance develops during idiophase.Resistance mechanisms include enzymatic mod-ification ofthe antibiotic, alteration ofthe target,and decreased uptake of excreted antibiotic.

Structural genes coding for antibiotic synthe-tases are usually chromosomal. However, in onecase these enzymes are coded by plasmid genes.Regulatory genes seem to be plasmid coded.Mechanisms that control the initiation of anti-biotic synthesis include repression and inhibitionof antibiotic synthetases. There is evidence thatin some cases the repression acts at the tran-scription level. In cases of branched pathwaysleading to a primary metabolite and an anti-biotic, the primary metabolite interferes withantibiotic formation by inhibiting an early stepof the common pathway, thus preventing accu-mulation of the antibiotic precursor.

Specific mechanisms regulating the onset ofantibiotic synthesis include carbon cataboliterepression, nitrogen metabolite regulation, phos-phate regulation, and induction. Cessation ofantibiotic biosynthesis occurs via decay of anti-biotic synthetases, feedback inhibition, andrepression of these enzymes.Routine strain improvement programs have

successfully yielded high-producing mutants ofantibiotic producers. Attempts at intentionalderegulation of such cultures have begun onlyrecently, but have been successful. Therefore,the antibiotic industry will increase its use ofrational screening techniques and feature moregenetic engineering of antibiotic-producingstrains, including the manipulation of plasmidscontaining genes involved in antibiotic forma-tion.

LITERATURE CITED1. Aharonowitz, Y. 1979. Regulatory interrela-

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2. Aharonowitz, Y., and A. L Demain. 1978.Carbon catabolite regulation of cephalsoporinproduction in Streptomyces clavuligerus. An-timicrob. Agents Chemother. 14:159-164.

3. Aharonowitz, Y., and A. L Demain. 1979.Nitrogen nutrition and regulation of cephalo-sporin production in Streptomyces clavulige-rus. Can. J. Microbiol. 25:61-67.

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