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110315 vancomycin

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    110315 vancomycin 110315 vancomycin Document Transcript

    • Vancomycin Production in Batch andContinuous CultureJames J. Mclntyre," Alan T. Bull, and Alan W. BunchResearch School of Biosciences, University of Kent, Canterbury, Kent CT27NJ, United KingdomReceived June 9, 1995/Accepted September 1 I , 1995Production of the glycopeptide antibiotic vancomycin by that are metabolically derived from tyrosine and acetate,two Amycolatopsis orientalis strains was examined in respectively.6 These amino acids are probably assem-batch shake flask culture in a semidefined medium with bled via a thiotemplate mechanism similar to that re-peptone as the nitrogen source. Different growth andproduction profiles were observed with the two strains; ported for other peptide antibiotics."specific producion (V,,,) was threefold higher with strain Despite the excellent progress on the genetic andATCC 19795 than with strain NCIMB 12945. A defined molecular biology of antibiotic biosynthesis, in severalmedium with amino acids as the nitrogen source was cases in Streptorny~es,~~~~~~~~~ there still remains poordeveloped by use of the Plackett-Burman statistical understanding of the physiological regulation and con-screening method. This technique identified certainamino acids (glycine, phenylafanine, tyrosine, and argi- trol of their production. The classical view that antibioticnine) that gave significant increased specific production, production in batch culture occurs once the growth ratewhereas phosphate was identified as inhibitory for high has declined as a consequence of nutrient limitation,"specific vancomycin production. Experiments made with is not always the case. Several examples of antibioticsthe improved medium and strain ATCC 19795 showed that are produced during exponential growth, especiallythat vancomycin production kinetics were either growthdissociated or growth associated, depending on the when chemically defined media are employed, haveamino acid concentration. In chemostat culture at a been r e p ~ r t e d . ~ ~ Until, now, ~ ~ ~ , " ~ ~ , experimental studiesconstant dilution rate (0.087 h-), specific vancomycin pro-on vancomycin production have focused attention onlyduction rate ( qvancomycln)decreased linearly as the me- on batch p r o c e s s e ~ .No ~ ~ ~ , research on continuous cul-dium phosphate concentration was increased from 2 to ture of A. orientalis has been reported.8 mM. In both phosphate and glucose limited chemo- stats, qvancomyclnfunction of specific growth rate; the was a Although the advantages of continuous culture for maximum value was observed at D = 0.087 h- (52% of the study of microbial product formation has been rec-t h e maximum specific growth rate). Under phosphate ognized for many years, very few studies on continuous limited growth conditions, qvanCOmyclnwas threefold higher antibiotic production have been r e p ~ r t e d . ~ Despite the(0.37 mglg dry weight/h) than under glucose limitation potential of increased volumetric productivity com- (0.12 mg/g dry weight/h). 0 1996 John Wiley & Sons, Inc. pared to batch culture, continuous culture processes Key words: Amycolatopsis orientalis vancomycin pro-duction * chemostat culture * phosphate inhibition have not been employed in commercial antibiotic pro- duction, primarily as a result of strain degeneration lead-INTRODUCTION ing to lower productivity. Nevertheless, the technique provides a useful means of researching the relationshipThe actinomycete Amycolatopsis orientalis (formerly between the physiological status of an organism andclassified as Nocardia and Streptomyces orientalis17)pro- the production of antibiotics under steady-state cultureduces the glycopeptide antibiotics vancomycin and N- conditions at defined, but varied, growth rates. Pub-demethylvancomycin.3.21 The glycopeptide family of an- lished data on the relationship between growth ratetibiotics have a highly specific mode of action against and antibiotic production in chemostat culture differ,Gram-positive bacteria binding to the terminal carboxyl depending on the strain and culture conditions used ingroup of the developing peptidoglycan polymer.26Van- the study. Antibiotic production has been reported tocomycin and related glycopeptides continue to be the be both positively and negatively correlated to increasedantibiotics of choice against P-lactam resistant entero- growth rate and controlled by the nature of the growthbacteria, although in recent years an increase in resis- limiting nutrient. to them has been reported.41Little information is In the present study, production of the glycopeptideavailable regarding vancomycin biosynthesis. Precursor vancomycin by A. orientalis strains ATCC 19795 andstudies have shown that the aromatic rings of the agly- NCIMB 12945 is compared in batch culture. Investiga-cone are composed of two unusual amino acids, p - tions in continuous culture over a range of specifichydroxyphenylglycine and 3,5-dihydroxyphenylglycine, growth rates and nutrient limitations in a defined me- dium, developed by a statistical screening design, is de- * To whom all correspondence should be addressed. scribed for A. orientalis strain ATCC 19795. Evidence isBiotechnology and Bioengineering, Vol. 49, Pp. 412-420 (1996)0 1996 John Wiley & Sons, Inc. CCC 0006-3592/96/040412-09
    • presented for the negative effects of increased inorganic 30°C for 4-5 days at 220 rpm. For fermenter cultures,phosphate on vancomycin production. a 5-L Electrolab reactor (working volume 3.35 L) was used. Agitation was controlled at 600 rpm and the con- tents mixed by two six-bladed disc-turbine impellersMATERIAL AND METHODS (55-mm diam.). An aeration rate of 1 vessel volume per minuteOrganisms and Culture Conditions (1vvm) was employed, and temperature was controlled at 30°C. The dissolved oxygen concentration (measuredAmycolatopsis orientalis strains (ATCC 19795 and with an Ingold polarographic electrode) was not con-NCIMB 12945) were maintained in a frozen vegetative trolled, but did not fall below 75% air saturation in thestate in 40% glycerol (v/v) in 2 mL cryopreservation fermenter studies reported. Constant culture volumevials at -80°C. Bacillus subtilis (ATCC 6633), the vanco- was maintained in batch culture by a water feed tomycin bioassay organism, was maintained as a spore compensate for sampling and evaporation. In continu-suspension. A complex seed medium containing (g/L) ous cultures steady states were assumed to have beenglucose (5), soluble starch (lo), peptone (Oxoid, 5 ) , and achieved after three fermenter volumes of medium hadyeast extract (Oxoid, 2) was used for preparation of the been replaced and the establishment of constant dryvegetative inoculum. The semidefined medium used to weight. Steady-state values of the measured parameterscompare the productivity of the two strains was modified were recorded as the mean (95% confidence limits) offrom that reported for the production of N-demethyl- nine readings per steady state (each measurement wasvancomycin3 and contained (g/L) glucose (20), peptone made at an interval of three fermenter volume changes).(Oxoid, 5), M g S 0 4 . 6 H 2 0 (0.75), NaCl (l),KCl (OS), Batch studies in shake flasks and Plackett-Burman ex-and 10 mL trace metal solution. Sucrose at 10 g/L re- periments show the mean data from triplicate experi-placed glucose in the media for strain NCIMB 12945, ments; fermenter batch studies report mean data frombecause in previous experiments it was shown not to duplicate experiments.produce vancomycin with glucose (unpublished data).The carbon sources were autoclaved separately. The defined medium developed from Plackett- AnalysisBurman analysis (see below) contained (g/L) glucose For the estimation of biomass dry weight, 10-mL sam-(25), NaCl (l),MgS04 7 H 2 0 (1.75), KH2P04 (0.27), ples of culture were centrifuged (at 11,OOOg for 10 min),L-asparagine (0.08), L-glutamate (0.4), L-aspartate the pellet was washed twice with distilled water, resus-(0.18), L-glycine (0.9), L-phenylalanine (0.07), L-argi- pended, added to a predried aluminum foil boat, andnine (0.25), L-tyrosine (O.l), L-leucine (0.125), and 20 dried at 90°C to constant weight. Vancomycin in culturemL trace metal solution. The trace metal solution con- filtrates was recovered by affinity chromatography usingtained (X100 strength) (g/L) FeS04 . 7 H 2 0 (l), n S 0 4 Z the cell wall ligand D-alanyl-D-ala~~ine.~~ The antibiotic. 7 H 2 0 (l), MnS04 . 4H20 (0.5), CaCI2 6 H 2 0 (0.2), concentration was measured by high-performance Iiq- +CuS04 . 5 H 2 0 (0.2), CoCI2 * 6 H 2 0 (O.l), and NaMo04 uid chromatography (HPLC) as r e p ~ r t e d ,except that ~. 2H20 (0.1). a mobile phase consisting of acetonitrile :methanol : am- In shake flask culture the p H was maintained at 6.7 monium acetate (0.2M) :water (45 : 10 : 10: 35 v/v) wasby the addition of MOPS (3-[N-morpholino]propane- used. A comparative microbiological assay using B.sulfonic acid) at 10 g/L. In fermenter cultures, pH was subtilis (ACTCC 6633) as the indicator organism wasmaintained at 6.8-7.0 by the automatic addition of 2M also used on a 36-well bioassay plate (Nunc). This assayHCl and 2M NaOH. Culture purity was checked by was shown to be linear over the range tested (1-90 pg/plating each strain onto Nutrient Agar (Oxoid) plates mL). Residual glucose was measured using a test kit(2.5% wlv) daily and incubating at 30°C overnight. Me- (GOD-Perid, Boehringer Mannheim), based on the glu-dia used in continuous culture experiments were pre- cose oxidase reaction. Residual phosphate was mea-pared by filter sterilization in 20-L batches. In separate sured using a colorimetric assay based on the formationchemostat experiments, phosphate and glucose were de- of phosphomolybdate by the reaction of phosphate andtermined to be the growth limiting nutrients, because ammonium m ~ l y b d a t eAmino acids were determined .~~on increasing the concentration of these nutrients, the by gradient HPLC as their o-phthaldehyde derivative^.^concentration of biomass increased c o r r e s p ~ n d i n g l y . ~ ~ ~ ~ Shake flask experiments used 250-mL, wide neckedflasks containing 40 mL of medium. Inoculum was pre- Statistical Screening Method: The Plackett-Burman Designpared from 48-h seed cultures grown in the complexmedium at 30°C on an orbital shaker (Infors AG, Swit- The Plackett-Burman design is a partial factorialzerland) at 220 rpm. Cells were harvested, washed twice method that allows the testing of multiple independentin physiological saline, and used to inoculate production process variables within a single e ~ p e r i m e n t .The de- ~flasks at 5% (viv). Production flasks were incubated at sign has been applied to the optimization of culture MclNTYRE, BULL, AND BUNCH: VANCOMYCIN PRODUCTION IN BATCH AND CONTINUOUS CULTURE 413
    • media for the production of primary and secondary me-tabolites in microbial and mammalian cell cultures andused in the optimization of commercially important en-zyme The design was used to develop The relationship between the variance of an effect anda suitable defined medium for studying vancomycin pro- the standard error (SE) of an effect is given by theduction in batch and chemostat culture. equation: All the experiments were carried out according to adesign matrix, which is based on the number of variables SEeff = G (3)to be studied. Each row represents one trial (culture Thus, it is possible to calculate the effect of the experi-medium) and each column represents a single variable mental variables and find the standard error (SE) of(medium component). Once the independent variables the effect from the effect of the dummy variables. Thehave been selected, they are examined at two levels significance of each variable can then be determined bydenoted high (+) and low (-) in each trial. A number using Student’s t test:of variables are designated as “dummy” variables be-cause no change is made to them, but they are used to (4)estimate the experimental error. Table I illustrates thematrix used in the two separate experiments employed The t test for an individual effect allows an evalutionin medium development. The response measured (van- of the probability of finding an effect. In our experi-comycin specific and volumetric productivities) was re- ments, only confidence levels above 80%were accepted.corded for each trial. Thus it was possible to rank the variables that signifi- Statistical analyses identified those medium variables cantly effect vancomycin synthesis either positively orthat had a significant effect on vancomycin specific pro- negatively, depending on the overall influence of theduction. The effect of a variable (En)on the response variable on the measured response.(R) is the difference between the average for the experi-ments at high and low values: RESULTS R at (+) - R at (-) En = (1) 4 4 . Vancomycin Production in Batch CultureOnce the dummy variables are selected, their effects Shake flask studies of the two A. orientalis strainsare calculated in the same way as the real variables. If NCIMB 12945 and ATCC 19795 grown in a semidefinedthere is no error in experimentation the effect of the medium with peptone as the nitrogen source revealeddummy variables will be close to zero. some differences between the two strains. Previous ex- The dummy variables are used to calculate the vari- periments concerned with optimizing the carbon sourceance of an effect (Veff): for production indicated that glucose and sucrose gave Table I. Plackett-Burman matrix used in study of 11 variables with 12 trials Variables Trial A B C D (E) (F) G (H) I J K 1 + + - + + + - - - + - 2 + - + + + - - - + - + 3 - + + + - - - + - + + 4 + + + - - - + - + + - 5 + + - - - + - + + - + 6 + - - - + - + + - + + I - - - + - + + - + + + 8 - - + - + + - + + + - 9 - + - + + - + + + - - 10 + - + + - + + + - - - 11 - + + - + + + - - - + 12 - - - - - - - - - - - The same design matrix was used in separate experiments in the optimization of a semide- fined medium and in the development of a defined medium containing amino acids. Variable A, glucoseltyrosine; B, peptonelasparagine; C , KCUarginine; D, NaCVleucine; G, MgS04 . 7H20/phenylalanine; I, KH2POJglycine, J, MOPYglutamate; K, trace metals/aspartate, in the two respective experiments. Variables in parenthesis (E, F, H) are dummy variables. (+) High level of the particular variable; (-) low level of the same variable.414 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 49, NO. 4, FEBRUARY 20, 1996
    • the highest vancomycin titers with strains ATCC 19795 In addition to their different vancomycin productionand NCIMB 12945, respectively (unpublished data). patterns, the two strains appeared to have different mor-With A. orientalis strain NCIMB 12945, vancomycin phological characteristics. When grown on nutrientcould be detected during biomass production; but the agar, A . orientalis strain ATCC 19795 produced whiteconcentration continued to increase after growth had aerial hyphae and sporulated after 7-10 days incubation.stopped (Fig. 1A). In contrast, vancomycin production In contrast, A. orientalis strain NCIMB 12945 producedby strain ATCC 19795 increased in parallel with the neither spores nor aerial hyphae under the same growthbiomass concentration decreasing as the culture entered conditions. Microscopic examination of samples fromthe stationary phase (Fig. 1B). Specific vancomycin pro- the respective cultures described in Figure 1. showedduction was threefold higher with ATCC 19795, and that strain ATCC 19795produced a fragmented pelletedover repeated experiments it was shown to be stable. growth form, whilst strain NCIMB 12945 grew in a fila-In contrast, production with strain NCIMB 12945 was mentous growth form showing no evidence of pelleting.found to be unstable over repeated experiments. There- To quantify the effects of nutrient stress on vancomy-fore, A. orientalis strain ATCC 19795 was selected for cin biosynthesis, a chemically defined medium was re-examining vancomycin production in batch and continu- quired. Shake flask experiments using the reported de-ous culture experiments. fined medium for vancomycin p r o d u c t i ~ with strain n~~~~ ATCC 19795 showed growth and antibiotic production to be erratic (unpublished data); consequently addi- 2 -40 tional medium development was undertaken. a 2 Initially the Plackett-Burman design was used to identify and optimize components of the semidefined m 1.55 -30 medium that were important in stimulating specific van- 3 comycin production. Further experiments examined the m m - P U - I F effects of amino acids (selected as potential precursors of vancomycin) on specific production. Statistical analy- 1.og - 2 0 g .- C u Y In sis of the data from the first experiment revealed that r peptone, MgS04 . 7H20, and trace metals solution had s 0.5g - 1 0 a positive (stimulatory) effect; phosphate (as KH2P04) > was identified as having a strongly negative (inhibitory) effect on specific vancomycin production (Table 11, col- umn A). In addition, the “dummy” variable (E) from 0 -0 the matrix were also identified as significant (Table IIA). 0 20 40 60 80 100 120 This variable was included in the calculation of the vari- ance as a measure of the experimental error. In the second experiment, the amino acids glycine, phenylalanine, tyrosine, and arginine were also identi- fied as positive variables. The amino acids asparagine - 100 and glutamate were identified as having a negative ef- - ~ 0 . 7- 5 - 6 fect; aspartate and the three “dummy” variables, had -80 f no effect at all on specific vancomycin production (Table 5- -E - E fn a2 11, column B). In Table I1 the sign of each calculated $ 0.50 - -60 8 effect was applied to its significance level merely to c: n - 3 depict direction of the effect. Using the results of these I : r (3 studies, a fully defined medium (see Material and Meth- P - 40 0.25 - ods) was formulated that gave specific production com- parable to that achieved previously with the semidefined - 20 medium. All further experiments reported refer to this medium modified as stated. 0- -0 Analysis of residual nutrients in experiments with the 0 20 40 60 80 100 120 defined medium indicated that growth was probably Tirne(h) (6) nitrogen limited. Residual glucose and phosphate, butFigure 1. Time course of shake flask batch culture o A. orienfulis f no amino acids, could be detected after 24-h cultivation,(A) strain NCIMB 12945 and (B) strain ATCC 19795 grown in a suggesting that growth had ceased due to nitrogen limi-semidefined medium with peptone as the nitrogen source. Sucrose tation. Therefore the amino acid concentration in thereplaced glucose as the carbon source for growth and production forstrain NCIMB 12945. (0) Biomass dry weight; (m)specific vancomy- medium was doubled and growth and vancomycin pro-cin production; ( 0 )residual phosphate; (0)residual sucrose (glucose duction profiles were re-examined. Figure 2A shows thein B). batch profile without and Figure 2B the profiles with MclNTYRE, BULL, AND BUNCH: VANCOMYCIN PRODUCTION IN BATCH AND CONTINUOUS CULTURE 415
    • 1Table 1 . Positive and negative medium components of semide- 1 120fined and defined media identified from statistical analysis of Plackett- 1.8Burman experiments. Significance - c1.4 1.6 100 I Medium Component level (%) i z - E 1.2 80 0 i zA Peptone +99 E MgS04’7H20 +YO J 1.0 m 60 .cSemidefined Trace metal solution +90 a 8 0.8 0 Dummy (E) +80 .c 3 KH2PO4 -99 n 0.6 . 40 NaCllKCl NS Glucose NS 0.4 20 “Dummy” (F,H) NS 0.2B Glycine + 99 0 0 Phenylalanine +95 0 20 40 60 80 100 120 Tyrosine + 80 Time(h) Arginine + 80 (a)Defined Glutamate -80 2 120 Asparagine - 80 (E Aspartate NS 1.75 Leucine NS 100 “Dummy” (E,F,H) NS Flasks were harvested after 96 h. Biomass concentration, pH, and - g 1.50 - 80 9vancomycin productivity were measured. Triplicate flasks were usedfor each trial. The sign of each calculated effect was applied to its - m 1.25 i z Esignificance level merely to depict the direction of the effect. Letters z r 1.00 60in parentheses denote the variable key in Table I. Only confidence P 2 0.75 slevels above 80% are accepted. NS, not significant for specific vanco- n 40 i?mycin productivity. 0.50 20 0.25increased amino acid concentration. Although vanco-mycin was produced during the growth phase with single 0 Dconcentration of amino acids, vancomycin concentra- 0 20 40 60 80 100 120tion increased after 48 h as the culture entered the Tirne(h)stationary phase (Fig. 2A). In contrast, in the medium (b) Figure 2. Time course of fermenter batch culture of A. orientaliswhere the amino acid concentration had doubled, van- strain ATCC 19795 in a defined medium with amino acids as thecomycin increased as biomass concentration increased nitrogen source at (A) single and (B) double concentrations. (0)and reached a maximum as the culture entered the sta- Biomass dry weight, (H)specific vancomycin production; ( 0 )residualtionary phase (Fig. 2B). The maximum specific growth phosphate; (0)residual glucose.rate (pmax) increased slightly from 0.14 to 0.17 h-l, withthe biomass concentration also increasing from 4.3 to5.9 g/L after the modification. defined media, it was possible to use the chemostat to It was shown previously that vancomycin production, evaluate the production of vancomycin under variousbut not growth, can be affected by the phosphate con- growth conditions. The negative effect of increasedcentration of the m e d i ~ m . In ?cultures where the ~ ~~ phosphate concentration on vancomycin productionamino acids concentration was doubled at the start of was investigated first. Phosphate in the incoming me-the incubation, there was a earlier depletion of phos- dium feed was varied from 2 to 8 mM at a constantphate (Fig. 2A, B). If the concentration of phosphate dilution rate ( D = 0.087 h-’). Figure 3 shows that aswas increased from 2 to 5 mMsuch that the cultures were the phosphate concentration increased, the steady-stateno longer phosphate depleted, vancomycin production specific vancomycin production rate ( qvancomycin) andcould no longer be detected, confirming the negative biomass concentration were greatly affected. In con-effect of phosphate seen in Plackett-Burman experi- trast, over the range of phosphate concentrations tested,ments. The additional phosphate resulted in a 5% in- the specific rate of glucose uptake ( qglucose) changedcrease in biomass levels (data not shown). much less dramatically. At the highest phosphate concentration testedVancomycin Production in Chemostat Culture (8 mM), qvancomycin fell to zero from a maximum ofThere have been no previous reports of vancomycin 0.43 mg/g dry weight/h when the incoming phosphateproduction in chemostat culture. Using the modified concentration was 2 mM. The maximum steady-state416 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 49, NO. 4, FEBRUARY 20, 1996
    • I.o 2 I0 - - 0.8 0 3 - E! f 1.5= z - E - i m m 2 0.6 3 Em E m c .- E I 1.0 2 0 s 2 7.5 2 0.4 r: v - - n m u) d u) 0.5; 0.2 ! i d 5 0 0 5 0 2 4 6 8 10 0 0.03 0.06 0.09 0.12 0.15 Phosphate Concentration (mM) Dilution Rate (h-)Figure 3. Chemostat culture of A. orientalis strain ATCC 19795 with Figure 4. Phosphate limited chemostat culture of A . orientalis strainvarying phosphate concentration in the feed medium. Dilution rate ATCC 19795: the effect of dilution rate. (A) Steady-state biomassD = 0.087 h-: ( A ) steady-state biomass dry weight; (W) qvancomycln; dry weight; (W) qvancamycin; (0) qglucose.(0)qglucose; )residual phosphate. (0 0.14 h-l. The steady-state biomass concentration wasbiomass concentration was attained with 4 mM phos- relatively constant for all except the highest dilutionphate. Analysis of residual nutrients from each steady rates used, where it decreased, whereas qglucose increasedstate showed that only those at the highest phosphate from 0.15 to 0.46 mmoVg dry weightlh before fallingconcentration (8 mM) had no detectable amino acids slightly at the highest dilution rate tested (Fig. 4). Thepresent, suggesting that, at this concentration of phos- residual steady-state phosphate concentration was closephate growth, probably was nitrogen limited. This result to zero over the range of dilution rates tested. A similaris supported by the decrease in qglucose biomass con- and profile was seen with the qvancomycin qglucose, and suggest-centration at 8 mM phosphate and was confirmed by ing a possible relationship between glucose uptake andpulsing the culture with additional amino acids and ob- vancomycin biosynthesis. This hypothesis was tested inserving an increase of 10% in biomass concentration glucose limited chemostat culture.(data not shown). Residual phosphate increased from Figure 5 shows data from a glucose limited chemostatzero at lower feed concentrations (below 4 mM) to a (glucose feed concentration at 10 g/L). As with phos-maximum of 1.7 mM at the highest medium concentra- phate limitation, qvancomycin increased with dilution alsotion tested (Fig. 3). In addition, acid production was rate reaching a maximum of 0.12 mg/g dry weight/h atobserved as the phosphate concentration in the incom- D = 0.087 h-l; this value was threefold lower than thating medium increased, an observation previously madein batch culture studies. The nature of the metabolites 1.6 i 0.15responsible for this metabolic change is unknown. Ex-cretion of organic acid metabolites resulting from anoverflow in carbon metabolism has been reported pre- -viously in Streptomyces sp. under conditions of nitrogenlimitation in which the major metabolites were identi- - 6 - f 1.2 - 0.12 c. - ffied as pyruvate and a-ketoglutarate. m 0.09 : Because the maximum qvancomycin obtained at the waslowest phosphate feed concentration, vancomycin pro- 0 - 0.8 - - E Eduction was examined over a range of dilution rates in 8 - (Y 0.06 6 0 < Echemostat culture to evaluate the effect of specific $0.4 - CTgrowth rate on vancomycin biosynthesis under condi- 0.03tions of phosphate limitation (phosphate concentrationin the feed medium was 1 mM). Dilution rates rangingfrom 8 to 80% of pmax (0.17 h-l) were selected. The 0maximum qvancc,mycin mg/g dry weight/h) was ob- (0.37 0 0.03 0.06 0.09 0.12 0.15tained at a dilution rate of 0.087 h- (52% of the pmax Dilution Rate (h-)of this medium) as shown in Figure 4. The steady-state Figure 5. Glucose limited chemostat culture of A. orientalis strainvancomycin concentration decreased from 90 to 13 pgl ATCC 19795; the effect of dilution rate. (A)Steady-state biomassmL as the dilution rate increased from D = 0.027 to dry weight; (m) qvancomycin; (0) qglucose. MclNTYRE, BULL, AND BUNCH: VANCOMYCIN PRODUCTION IN BATCH AND CONTINUOUS CULTURE 417
    • observed in phosphate limited chemostats, at a similar fector of vancomycin synthesis, Table 11) are involvedgrowth rate. The vancomycin steady-state concentration in biological energy metab01ism.l~dropped from 18 to 1 pg/mL as the dilution rate was The growth of Klebsiella aerogenes under carbon, ni-increased from 0.027 to 0.14 h-’. As expected qglucose trogen, or phosphate limitation reveals large differencesincreased as dilution rate increased, reaching a maxi- in the biomass produced in batch cultures and themum rate threefold higher than observed in phosphate growth rates attained.15 Similar studies on the produc-limited cultures (Fig. 5). Residual steady-state glucose tion of erythromycin by Saccharopolysporu erythraea,levels remained close to zero as the dilution rate was in- showed production and growth patterns to differ, de-creased. pending on the growth limiting nutrient.22 However, Under glucose limitation, a low vancomycin producer during growth and antibiotic biosynthesis, there is amay have been selected in prolonged chemostat culture, continuous exchange of precursors and energy betweenand this could explain the observed differences in vanco- primary and secondary pathways, some of which maymycin specific production rate to that obtained under be rate limiting. Thus, if precursor supply from primaryphosphate limitation. Decreases in antibiotic production pathways is increased, a growth associated pattern mightunder various nutrient and growth conditions in contin- be expected. This integration of metabolic activities mayuous culture studies have frequently been reported for explain the growth associated production pattern seenS t r e p t ~ r n y c e s . ~ ~Therefore, ~ biomass was taken ,~~,~~, ~ on doubling the amino acid concentration in the definedfrom the chemostat after 500 h and the growth and medium in batch culture.vancomycin productivities assessed in batch culture. Re- Recently published studies with strain ATCC 19795insults from these experiments showed no significant dif- batch culture using a defined medium containing glucoseferences in the batch culture characteristics of the chem- and sodium nitrate as carbon and nitrogen source, re-ostat isolate and the original culture shown in Figure 2B. ported vancomycin production to be growth dissociated and only beginning at the onset of glucose depletion from the medium.6 However, in the studies reportedDISCUSSION here, this was not the case. The use of chemostat culture confirmed that vanco-This study has shown major physiological differences mycin production was regulated by the medium phos-between the two vancomycin producing strains that phate concentration. Phosphate and carbon catabolitewere investigated. The strains ATCC 19795and NCIMB control of antibiotic biosynthesis occurs at the transcrip-12945 are reported in the literature as being the same tional level, although inhibition of specific synthetic en-isolate.” However, they were distinguishable morpho- zymes may also occur.yR Phosphate control sequenceslogically when grown both on agar and in submerged similar to the “phosphate box” of Escherichia coli haveculture and showed different vancomycin production been reported in many of the antibiotic producing Strep-patterns. Given the possible relationship between anti- tomycetes in~estigated.’~ intracellular effector that Thebiotic production and cellular differentiation, it is not mediates phosphate control via a binding protein issurprising that the two strains had different vancomycin thought to be a highly phosphorylated nucleotide thatproduction characteristics.20 Other studies have com- acts as a sensor of the phosphate level in the culturepared antibiotic production with respect to cell mor- medium.18 This type of regulation could explain thephology where pellet formation has been shown to give immediate decrease observed in vancomycin productionrise to different growth and production kinetics, de- when increasing the medium phosphate content inpending on the size and diffusion characteristics of the chemostat culture (Fig. 3).pellet when compared to a filamentous well-dispersed Production of the antibiotics nikkomycin and juglo-culture.40In the experiments reported, no attempt was mycin by Streptomyces tendae are tightly regulated bymade to correlate morphology with vancomycin produc- the phosphate concentration of the medium: high phos-tion with the two strains, because this was not the aim phate stimulates juglomycin and decreases nikkomycinof this present work. It is difficult to assess whether this production and the opposite occurs when the concentra-difference in growth and production pattern stems from tion of phosphate is lowered.38In both phosphate andthe different carbon sources used in the experiments. glucose limited chemostat culture, specific growth rate In our development of a defined medium, the is another important factor regulating vancomycin pro-Plackett-Burman design identified the nitrogen source duction; qvancomycin increased as growth rate increased(peptone or amino acids) as significant for improved to 0.087 h-’ before decreasing at the highest growth ratespecific vancomycin production (Table 11). Given the tested. The imposition of phosphate limitation mini-structure of vancomycin and the few reported precursor mized the repressive effect of phosphate on production,studies on its biosynthesis, the positive effect of peptone, and qvancomycin threefold higher than that under glu- wasglycine, phenylalanine, arginine, and tyrosine on specific cose limitation.production may relate to their role in precursor supply.16 Similar studies in chemostat culture have revealedPhosphate (a negative) and magnesium (a positive ef- maximal specific production of macrolide and aminogly-418 B I O T E C H N O L O G Y A N D BIOENGINEERING, VOL. 49, N O . 4, FEBRUARY 20, 1996
    • coside antibiotics under phosphate limited g r o ~ t h . ~ , 12.~James, P. D., Edwards, C., Dawson, M. 1991. The effects of tem- ~ ,~~In these chemostat cultures biomass concentration de- perature, pH and growth rate on secondary matabolism in Strepto- rnyces thermoviolaceus grown in chemostat culture. J. Gen. Micro-clined dramatically at the highest growth rates tested, biol. 137 1715-1720.suggesting that A. orientalis was unable to sustain 13. Jehl, F., Gallion, C. Montail, H. 1987. Determination of vancomy-a constant growth efficiency and vancomycin biosyn- cin in human serum by high pressure liquid chromatography.thesis. Antimicrob. Agents Chemother. 2 7 503-507. Results from these chemostat experiments has high- 14. Kirby, R. 1992. The isolation and characteristics of antibiotic biosynthesis genes. Biotechnol. Adv. 10 561-576.lighted several important physiological factors regulat- 15. Kason, A. C., Egli, T. 1993. Dynamics of microbial growth in theing vancomycin biosynthesis by A . orientalis strain deceleration and stationary phase of batch culture, pp. 103-ATCC 19795 in a defined medium. Information gained 120. In: S. Kjelleberg (ed), Starvation in bacteria. Plenum Press,from these studies is enabling the physiological condi- New York.tions controlling vancomycin production to be further 16. Lancini, G. C. 1989. Fermentation and biosynthesis of glycopep- tide antibiotics. Prog. Ind. Microbiol. 2 7 283-296.investigated in high cell density cultures established in 17. Lechevalier, M. P., Prauser, H., Labeda, D. P., Raun, J. S. 1986.chemostats employing biomass recycling. Results of Two new genera of Actinomycetes: Amycolatu gen. nov. andthese investigations will be reported in a later publi- Amycolutopsis gen. nov. Int. J. Syst. Bacteriol. 2 4 29-37.cation. 18. Liras, P., Asturias, J., Martin J. F. 1990. 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