110315 vancomycin

Vancomycin Production in Batch and Continuous Culture

James J. Mclntyre," Alan T. Bull, and Alan W. Bunch Research School of Biosciences, University of Kent, Canterbury, Kent CT2 7NJ, United Kingdom

Received June 9, 1995/Accepted September 1 I , 1995

Production of the glycopeptide antibiotic vancomycin by two Amycolatopsis orientalis strains was examined in batch shake flask culture in a semidefined medium with peptone as the nitrogen source. Different growth and production profiles were observed with the two strains; specific producion (V,,,) was threefold higher with strain ATCC 19795 than with strain NCIMB 12945. A defined medium with amino acids as the nitrogen source was developed by use of the Plackett-Burman statistical screening method. This technique identified certain amino acids (glycine, phenylafanine, tyrosine, and arginine) that gave significant increased specific production, whereas phosphate was identified as inhibitory for high specific vancomycin production. Experiments made with the improved medium and strain ATCC 19795 showed that vancomycin production kinetics were either growth dissociated or growth associated, depending on the amino acid concentration. In chemostat culture at a constant dilution rate (0.087 h-'), specific vancomycin production rate ( qvancomycln) decreased linearly as the medium phosphate concentration was increased from 2 to 8 mM. In both phosphate and glucose limited chemostats, qvancomycln was a function of specific growth rate; the maximum value was observed at D = 0.087 h-' (52% of the maximum specific growth rate). Under phosphate limited growth conditions, qvanCOmycln was threefold higher (0.37 mglg dry weight/h) than under glucose limitation (0.12 mg/g dry weight/h). 0 1996 John Wiley & Sons, Inc. Key words: Amycolatopsis orientalis vancomycin production * chemostat culture * phosphate inhibition

INTRODUCTION The actinomycete Amycolatopsis orientalis (formerly classified as Nocardia and Streptomyces orientalis17) pro- duces the glycopeptide antibiotics vancomycin and N- demethylvancomycin.3.21 The glycopeptide family of antibiotics have a highly specific mode of action against Gram-positive bacteria binding to the terminal carboxyl group of the developing peptidoglycan polymer.26 Van- comycin and related glycopeptides continue to be the antibiotics of choice against P-lactam resistant entero- bacteria, although in recent years an increase in resistance to them has been reported.41 Little information is available regarding vancomycin biosynthesis. Precursor studies have shown that the aromatic rings of the agly- cone are composed of two unusual amino acids, p - hydroxyphenylglycine and 3,5-dihydroxyphenylglycine,

* To whom all correspondence should be addressed.

that are metabolically derived from tyrosine and acetate, respectively.'6 These amino acids are probably assem- bled via a thiotemplate mechanism similar to that reported for other peptide antibiotics."

Despite the excellent progress on the genetic and molecular biology of antibiotic biosynthesis, in several cases in Streptorny~es,'~~'~~~~~~~ there still remains poor understanding of the physiological regulation and control of their production. The classical view that antibiotic production in batch culture occurs once the growth rate has declined as a consequence of nutrient limitation," is not always the case. Several examples of antibiotics that are produced during exponential growth, especially when chemically defined media are employed, have been r e p ~ r t e d . ~ ~ ~ , " , ~ ~ , ~ ~ Until now, experimental studies on vancomycin production have focused attention only on batch p r o c e s s e ~ . ~ , ~ ~ No research on continuous culture of A. orientalis has been reported.

Although the advantages of continuous culture for the study of microbial product formation has been rec- ognized for many years, very few studies on continuous antibiotic production have been r ep~r t ed .~ ' Despite the potential of increased volumetric productivity compared to batch culture, continuous culture processes have not been employed in commercial antibiotic production, primarily as a result of strain degeneration lead- ing to lower productivity. Nevertheless, the technique provides a useful means of researching the relationship between the physiological status of an organism and the production of antibiotics under steady-state culture conditions at defined, but varied, growth rates. Pub- lished data on the relationship between growth rate and antibiotic production in chemostat culture differ, depending on the strain and culture conditions used in the study. Antibiotic production has been reported to be both positively and negatively correlated to increased growth rate and controlled by the nature of the growth

In the present study, production of the glycopeptide vancomycin by A. orientalis strains ATCC 19795 and NCIMB 12945 is compared in batch culture. Investiga- tions in continuous culture over a range of specific growth rates and nutrient limitations in a defined medium, developed by a statistical screening design, is described for A. orientalis strain ATCC 19795. Evidence is

limiting nutrient.2.8.12.32.34.38

Biotechnology 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 phosphate on vancomycin production.

MATERIAL AND METHODS

Organisms and Culture Conditions

Amycolatopsis orientalis strains (ATCC 19795 and NCIMB 12945) were maintained in a frozen vegetative state in 40% glycerol (v/v) in 2 mL cryopreservation vials at -80°C. Bacillus subtilis (ATCC 6633), the vancomycin bioassay organism, was maintained as a spore suspension. A complex seed medium containing (g/L) glucose (5), soluble starch (lo), peptone (Oxoid, 5) , and yeast extract (Oxoid, 2) was used for preparation of the vegetative inoculum. The semidefined medium used to compare the productivity of the two strains was modified from that reported for the production of N-demethyl- vancomycin3 and contained (g/L) glucose (20), peptone (Oxoid, 5), MgS04. 6H20 (0.75), NaCl (l), KCl ( O S ) , and 10 mL trace metal solution. Sucrose at 10 g/L replaced glucose in the media for strain NCIMB 12945, because in previous experiments it was shown not to produce vancomycin with glucose (unpublished data). The carbon sources were autoclaved separately.

The defined medium developed from Plackett- Burman analysis (see below) contained (g/L) glucose (25), NaCl (l), MgS04 7H20 (1.75), KH2P04 (0.27), L-asparagine (0.08), L-glutamate (0.4), L-aspartate (0.18), L-glycine (0.9), L-phenylalanine (0.07), L-arginine (0.25), L-tyrosine (O. l ) , L-leucine (0.125), and 20 mL trace metal solution. The trace metal solution contained (X100 strength) (g/L) FeS04 . 7 H 2 0 (l), ZnS04 . 7H20 (l), MnS04 . 4H20 (0.5), CaCI2 + 6H20 (0.2), CuS04 . 5H20 (0.2), CoCI2 * 6H20 (O.l), and NaMo04

In shake flask culture the pH was maintained at 6.7 by the addition of MOPS (3-[N-morpholino]propane- sulfonic acid) at 10 g/L. In fermenter cultures, pH was maintained at 6.8-7.0 by the automatic addition of 2M HCl and 2M NaOH. Culture purity was checked by plating each strain onto Nutrient Agar (Oxoid) plates (2.5% wlv) daily and incubating at 30°C overnight. Me- dia used in continuous culture experiments were pre- pared by filter sterilization in 20-L batches. In separate chemostat experiments, phosphate and glucose were determined to be the growth limiting nutrients, because on increasing the concentration of these nutrients, the concentration of biomass increased co r re sp~nd ing ly . '~~~~

Shake flask experiments used 250-mL, wide necked flasks containing 40 mL of medium. Inoculum was pre- pared from 48-h seed cultures grown in the complex medium at 30°C on an orbital shaker (Infors AG, Swit- zerland) at 220 rpm. Cells were harvested, washed twice in physiological saline, and used to inoculate production flasks at 5% (viv). Production flasks were incubated at

. 2H20 (0.1).

30°C for 4-5 days at 220 rpm. For fermenter cultures, 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 impellers (55-mm diam.).

An aeration rate of 1 vessel volume per minute (1 vvm) was employed, and temperature was controlled at 30°C. The dissolved oxygen concentration (measured with an Ingold polarographic electrode) was not controlled, but did not fall below 75% air saturation in the fermenter studies reported. Constant culture volume was maintained in batch culture by a water feed to compensate for sampling and evaporation. In continuous cultures steady states were assumed to have been achieved after three fermenter volumes of medium had been replaced and the establishment of constant dry weight. Steady-state values of the measured parameters were recorded as the mean (95% confidence limits) of nine readings per steady state (each measurement was made at an interval of three fermenter volume changes). Batch studies in shake flasks and Plackett-Burman experiments show the mean data from triplicate experiments; fermenter batch studies report mean data from duplicate experiments.

Analysis

For the estimation of biomass dry weight, 10-mL samples of culture were centrifuged (at 11,OOOg for 10 min), the pellet was washed twice with distilled water, resus- pended, added to a predried aluminum foil boat, and dried at 90°C to constant weight. Vancomycin in culture filtrates was recovered by affinity chromatography using the cell wall ligand D-alanyl-D-ala~~ine.~~ The antibiotic concentration was measured by high-performance Iiq- uid chromatography (HPLC) as r e p ~ r t e d , ' ~ except that a mobile phase consisting of acetonitrile : methanol : ammonium acetate (0.2M) : water (45 : 10 : 10 : 35 v/v) was used. A comparative microbiological assay' using B. subtilis (ACTCC 6633) as the indicator organism was also used on a 36-well bioassay plate (Nunc). This assay was shown to be linear over the range tested (1-90 pg/ mL). Residual glucose was measured using a test kit (GOD-Perid, Boehringer Mannheim), based on the glucose oxidase reaction. Residual phosphate was measured using a colorimetric assay based on the formation of phosphomolybdate by the reaction of phosphate and ammonium m ~ l y b d a t e . ~ ~ Amino acids were determined by gradient HPLC as their o-phthaldehyde derivative^.^

Statistical Screening Method: The Plackett-Burman Design

The Plackett-Burman design is a partial factorial method that allows the testing of multiple independent process variables within a single e~per iment .~ ' The design 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 metabolites in microbial and mammalian cell cultures and used in the optimization of commercially important enzyme The design was used to develop a suitable defined medium for studying vancomycin production in batch and chemostat culture.

All the experiments were carried out according to a design matrix, which is based on the number of variables to be studied. Each row represents one trial (culture medium) and each column represents a single variable (medium component). Once the independent variables have been selected, they are examined at two levels denoted high (+) and low (-) in each trial. A number of variables are designated as “dummy” variables because no change is made to them, but they are used to estimate the experimental error. Table I illustrates the matrix used in the two separate experiments employed in medium development. The response measured (vancomycin specific and volumetric productivities) was recorded for each trial.

Statistical analyses identified those medium variables that had a significant effect on vancomycin specific production. The effect of a variable (En) on the response (R) is the difference between the average for the experiments at high and low values:

The relationship between the variance of an effect and the standard error (SE) of an effect is given by the equation:

SEeff = G (3)

Thus, it is possible to calculate the effect of the experimental variables and find the standard error (SE) of the effect from the effect of the dummy variables. The significance of each variable can then be determined by using Student’s t test:

(4)

The t test for an individual effect allows an evalution of the probability of finding an effect. In our experiments, only confidence levels above 80% were accepted. Thus it was possible to rank the variables that signifi- cantly effect vancomycin synthesis either positively or negatively, depending on the overall influence of the variable on the measured response.

RESULTS

Vancomycin Production in Batch Culture

Shake flask studies of the two A. orientalis strains NCIMB 12945 and ATCC 19795 grown in a semidefined medium with peptone as the nitrogen source revealed some differences between the two strains. Previous experiments concerned with optimizing the carbon source for production indicated that glucose and sucrose gave

(1) R at (+) - R at (-)

4 4 . En =

Once the dummy variables are selected, their effects are calculated in the same way as the real variables. If there is no error in experimentation the effect of the dummy variables will be close to zero.

The dummy variables are used to calculate the variance of an effect (Veff):

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 semidefined 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 and NCIMB 12945, respectively (unpublished data). With A. orientalis strain NCIMB 12945, vancomycin could be detected during biomass production; but the concentration continued to increase after growth had stopped (Fig. 1A). In contrast, vancomycin production by strain ATCC 19795 increased in parallel with the biomass concentration decreasing as the culture entered the stationary phase (Fig. 1B). Specific vancomycin production was threefold higher with ATCC 19795, and over repeated experiments it was shown to be stable. In contrast, production with strain NCIMB 12945 was found to be unstable over repeated experiments. There- fore, A. orientalis strain ATCC 19795 was selected for examining vancomycin production in batch and continuous culture experiments.

2

a 2 m

1.55 3

U - 1.og

.- u r s 0.5g

>

0

-40

-30

P F m - m I

- 2 0 g C Y

In

- 1 0

-0 0 20 40 60 80 100 120

~ 0 . 7 5 -

E

$ 0.50 - n

- 5- - c:

I: r P

0.25 -

0-

- 100

- 6 -80 f

E

-60 8 (3

- a2 fn

3 - - 40

- 20

-0 0 20 40 60 80 100 120

Tirne(h) (6)

Figure 1. Time course of shake flask batch culture of A. orienfulis (A) strain NCIMB 12945 and (B) strain ATCC 19795 grown in a semidefined medium with peptone as the nitrogen source. Sucrose replaced glucose as the carbon source for growth and production for strain NCIMB 12945. (0) Biomass dry weight; (m) specific vancomycin production; (0) residual phosphate; (0) residual sucrose (glucose in B).

In addition to their different vancomycin production patterns, the two strains appeared to have different mor- phological characteristics. When grown on nutrient agar, A. orientalis strain ATCC 19795 produced white aerial hyphae and sporulated after 7-10 days incubation. In contrast, A. orientalis strain NCIMB 12945 produced neither spores nor aerial hyphae under the same growth conditions. Microscopic examination of samples from the respective cultures described in Figure 1. showed that strain ATCC 19795 produced a fragmented pelleted growth form, whilst strain NCIMB 12945 grew in a filamentous growth form showing no evidence of pelleting.

To quantify the effects of nutrient stress on vancomycin biosynthesis, a chemically defined medium was re- quired. Shake flask experiments using the reported defined medium for vancomycin p r o d u c t i ~ n ~ ~ ~ ~ with strain ATCC 19795 showed growth and antibiotic production to be erratic (unpublished data); consequently additional medium development was undertaken.

Initially the Plackett-Burman design was used to identify and optimize components of the semidefined medium that were important in stimulating specific vancomycin production. Further experiments examined the effects of amino acids (selected as potential precursors of vancomycin) on specific production. Statistical analysis of the data from the first experiment revealed that peptone, MgS04 . 7H20, and trace metals solution had a positive (stimulatory) effect; phosphate (as KH2P04) was identified as having a strongly negative (inhibitory) effect on specific vancomycin production (Table 11, column A). In addition, the “dummy” variable (E) from the matrix were also identified as significant (Table IIA). This variable was included in the calculation of the variance as a measure of the experimental error.

In the second experiment, the amino acids glycine, phenylalanine, tyrosine, and arginine were also identified as positive variables. The amino acids asparagine and glutamate were identified as having a negative effect; aspartate and the three “dummy” variables, had no effect at all on specific vancomycin production (Table 11, column B). In Table I1 the sign of each calculated effect was applied to its significance level merely to depict direction of the effect. Using the results of these studies, a fully defined medium (see Material and Meth- ods) was formulated that gave specific production com- parable to that achieved previously with the semidefined medium. All further experiments reported refer to this medium modified as stated.

Analysis of residual nutrients in experiments with the defined medium indicated that growth was probably nitrogen limited. Residual glucose and phosphate, but no amino acids, could be detected after 24-h cultivation, suggesting that growth had ceased due to nitrogen limitation. Therefore the amino acid concentration in the medium was doubled and growth and vancomycin production profiles were re-examined. Figure 2A shows the batch profile without and Figure 2B the profiles with


B

Defined

Table 11. Positive and negative medium components of semidefined and defined media identified from statistical analysis of Plackett- Burman experiments.

Significance Medium Component level (%)

A Peptone + 99 MgS04’7H20 +YO

Semidefined Trace metal solution + 90 Dummy (E) + 80 KH2PO4 - 99 NaCllKCl NS Glucose NS “Dummy” (F,H) NS

Glycine Phenylalanine Tyrosine Arginine Glutamate Asparagine Aspartate Leucine “Dummy” (E,F,H)

+ 99 +95 + 80 + 80 - 80 - 80 NS NS NS

Flasks were harvested after 96 h. Biomass concentration, pH, and vancomycin productivity were measured. Triplicate flasks were used for each trial. The sign of each calculated effect was applied to its significance level merely to depict the direction of the effect. Letters in parentheses denote the variable key in Table I. Only confidence levels above 80% are accepted. NS, not significant for specific vancomycin productivity.

increased amino acid concentration. Although vancomycin was produced during the growth phase with single concentration of amino acids, vancomycin concentration increased after 48 h as the culture entered the stationary phase (Fig. 2A). In contrast, in the medium where the amino acid concentration had doubled, vancomycin increased as biomass concentration increased and reached a maximum as the culture entered the stationary phase (Fig. 2B). The maximum specific growth rate (pmax) increased slightly from 0.14 to 0.17 h-l, with the biomass concentration also increasing from 4.3 to 5.9 g/L after the modification.

It was shown previously that vancomycin production, but not growth, can be affected by the phosphate concentration of the m e d i ~ m . ~ ? ~ ~ In cultures where the amino acids concentration was doubled at the start of the incubation, there was a earlier depletion of phosphate (Fig. 2A, B). If the concentration of phosphate was increased from 2 to 5 mMsuch that the cultures were no longer phosphate depleted, vancomycin production could no longer be detected, confirming the negative effect of phosphate seen in Plackett-Burman experiments. The additional phosphate resulted in a 5% increase in biomass levels (data not shown).

Vancomycin Production in Chemostat Culture

There have been no previous reports of vancomycin production in chemostat culture. Using the modified

1

1.8

1.6

c1.4

E 1.2

J m 1.0 a 8 0.8 n. 0.6

0.4

0.2

0

- iz - .c

.c

2

1.75

g 1.50

1.25

z 1.00

- - - m r P

2 0.75 n

0.50

0.25

0

0 20 40 60 80 100 120 Time(h)

(a)

(E

120

100

I

80 0 E iz

60 0

3 40

20

0

120

100

80 9 E iz

s 60

40 i?

20

D 0 20 40 60 80 100 120

Tirne(h) (b)

Figure 2. Time course of fermenter batch culture of A. orientalis strain ATCC 19795 in a defined medium with amino acids as the nitrogen source at (A) single and (B) double concentrations. (0) Biomass dry weight, (H) specific vancomycin production; (0) residual phosphate; (0) residual glucose.

defined media, it was possible to use the chemostat to evaluate the production of vancomycin under various growth conditions. The negative effect of increased phosphate concentration on vancomycin production was investigated first. Phosphate in the incoming medium feed was varied from 2 to 8 mM at a constant dilution rate ( D = 0.087 h-’). Figure 3 shows that as the phosphate concentration increased, the steady-state specific vancomycin production rate ( qvancomycin) and biomass concentration were greatly affected. In contrast, over the range of phosphate concentrations tested, the specific rate of glucose uptake ( qglucose) changed much less dramatically.

At the highest phosphate concentration tested (8 mM), qvancomycin fell to zero from a maximum of 0.43 mg/g dry weight/h when the incoming phosphate concentration was 2 mM. The maximum steady-state


I .o

- 0.8 f m 2 0.6 E E

E! -

I

2 0.4 - d

0.2

0

2

- 0

1.5= z E 3 - m c

0 r:

m

1.0 2 n -

0.5;

d

0 0 2 4 6 8 10 Phosphate Concentration (mM)

Figure 3. Chemostat culture of A. orientalis strain ATCC 19795 with varying phosphate concentration in the feed medium. Dilution rate D = 0.087 h-': (A) steady-state biomass dry weight; (W) qvancomycln; (0) qglucose; (0) residual phosphate.

biomass concentration was attained with 4 mM phosphate. Analysis of residual nutrients from each steady state showed that only those at the highest phosphate concentration (8 mM) had no detectable amino acids present, suggesting that, at this concentration of phosphate growth, probably was nitrogen limited. This result is supported by the decrease in qglucose and biomass concentration at 8 mM phosphate and was confirmed by pulsing the culture with additional amino acids and ob- serving an increase of 10% in biomass concentration (data not shown). Residual phosphate increased from zero at lower feed concentrations (below 4 mM) to a maximum of 1.7 mM at the highest medium concentration tested (Fig. 3). In addition, acid production was observed as the phosphate concentration in the incoming medium increased, an observation previously made in batch culture studies. The nature of the metabolites responsible for this metabolic change is unknown. Ex- cretion of organic acid metabolites resulting from an overflow in carbon metabolism has been reported previously in Streptomyces sp. under conditions of nitrogen limitation in which the major metabolites were identified as pyruvate and a-ketoglutarate.'

Because the maximum qvancomycin was obtained at the lowest phosphate feed concentration, vancomycin production was examined over a range of dilution rates in chemostat culture to evaluate the effect of specific growth rate on vancomycin biosynthesis under conditions of phosphate limitation (phosphate concentration in the feed medium was 1 mM). Dilution rates ranging from 8 to 80% of pmax (0.17 h-l) were selected. The maximum qvancc,mycin (0.37 mg/g dry weight/h) was obtained at a dilution rate of 0.087 h-' (52% of the pmax of this medium) as shown in Figure 4. The steady-state vancomycin concentration decreased from 90 to 13 pgl mL as the dilution rate increased from D = 0.027 to

I 0

3 i m

E m

2

- .- s 7.5

v u) u)

!i 5

5 0 0.03 0.06 0.09 0.12 0.15

Dilution Rate (h-')

Figure 4. Phosphate limited chemostat culture of A. orientalis strain ATCC 19795: the effect of dilution rate. (A) Steady-state biomass dry weight; (W) qvancamycin; (0) qglucose.

0.14 h-l. The steady-state biomass concentration was relatively constant for all except the highest dilution rates used, where it decreased, whereas qglucose increased from 0.15 to 0.46 mmoVg dry weightlh before falling slightly at the highest dilution rate tested (Fig. 4). The residual steady-state phosphate concentration was close to zero over the range of dilution rates tested. A similar profile was seen with the qvancomycin and qglucose, suggesting a possible relationship between glucose uptake and vancomycin biosynthesis. This hypothesis was tested in glucose limited chemostat culture.

Figure 5 shows data from a glucose limited chemostat (glucose feed concentration at 10 g/L). As with phosphate limitation, qvancomycin also increased with dilution rate reaching a maximum of 0.12 mg/g dry weight/h at D = 0.087 h-l; this value was threefold lower than that

1.6 i

6 1.2 - - - f m : 0

0.8 - - (Y

8 - $0.4 -

0.15

0.12 - f

E

c. - 0.09 -

E 0

0.06 6 E <

CT

0.03

0 0 0.03 0.06 0.09 0.12 0.15

Dilution Rate (h-')

Figure 5. Glucose limited chemostat culture of A. orientalis strain ATCC 19795; the effect of dilution rate. (A) Steady-state biomass dry weight; (m) qvancomycin; (0) qglucose.


observed in phosphate limited chemostats, at a similar growth rate. The vancomycin steady-state concentration dropped from 18 to 1 pg/mL as the dilution rate was increased from 0.027 to 0.14 h-’. As expected qglucose increased as dilution rate increased, reaching a maximum rate threefold higher than observed in phosphate limited cultures (Fig. 5). Residual steady-state glucose levels remained close to zero as the dilution rate was increased.

Under glucose limitation, a low vancomycin producer may have been selected in prolonged chemostat culture, and this could explain the observed differences in vancomycin specific production rate to that obtained under phosphate limitation. Decreases in antibiotic production under various nutrient and growth conditions in continuous culture studies have frequently been reported for S t r e p t ~ r n y c e s . ~ ~ , ~ ~ , ~ ~ , ~ ~ Therefore, biomass was taken from the chemostat after 500 h and the growth and vancomycin productivities assessed in batch culture. Re- sults from these experiments showed no significant differences in the batch culture characteristics of the chemostat isolate and the original culture shown in Figure 2B.

DISCUSSION

This study has shown major physiological differences between the two vancomycin producing strains that were investigated. The strains ATCC 19795 and NCIMB 12945 are reported in the literature as being the same isolate.” However, they were distinguishable morpho- logically when grown both on agar and in submerged culture and showed different vancomycin production patterns. Given the possible relationship between antibiotic production and cellular differentiation, it is not surprising that the two strains had different vancomycin production characteristics.20 Other studies have compared antibiotic production with respect to cell morphology where pellet formation has been shown to give rise to different growth and production kinetics, depending on the size and diffusion characteristics of the pellet when compared to a filamentous well-dispersed culture.40 In the experiments reported, no attempt was made to correlate morphology with vancomycin production with the two strains, because this was not the aim of this present work. It is difficult to assess whether this difference in growth and production pattern stems from the different carbon sources used in the experiments.

In our development of a defined medium, the Plackett-Burman design identified the nitrogen source (peptone or amino acids) as significant for improved specific vancomycin production (Table 11). Given the structure of vancomycin and the few reported precursor studies on its biosynthesis, the positive effect of peptone, glycine, phenylalanine, arginine, and tyrosine on specific production may relate to their role in precursor supply.16 Phosphate (a negative) and magnesium (a positive ef-

fector of vancomycin synthesis, Table 11) are involved in biological energy metab01ism.l~

The growth of Klebsiella aerogenes under carbon, nitrogen, or phosphate limitation reveals large differences in the biomass produced in batch cultures and the growth rates attained.15 Similar studies on the production of erythromycin by Saccharopolysporu erythraea, showed production and growth patterns to differ, depending on the growth limiting nutrient.22 However, during growth and antibiotic biosynthesis, there is a continuous exchange of precursors and energy between primary and secondary pathways, some of which may be rate limiting. Thus, if precursor supply from primary pathways is increased, a growth associated pattern might be expected. This integration of metabolic activities may explain the growth associated production pattern seen on doubling the amino acid concentration in the defined medium in batch culture.

Recently published studies with strain ATCC 19795 in batch culture using a defined medium containing glucose and sodium nitrate as carbon and nitrogen source, reported vancomycin production to be growth dissociated and only beginning at the onset of glucose depletion from the medium.6 However, in the studies reported here, this was not the case.

The use of chemostat culture confirmed that vancomycin production was regulated by the medium phosphate concentration. Phosphate and carbon catabolite control of antibiotic biosynthesis occurs at the transcriptional level, although inhibition of specific synthetic en- zymes may also occur.yR Phosphate control sequences similar to the “phosphate box” of Escherichia coli have been reported in many of the antibiotic producing Strep- tomycetes in~estigated.’~ The intracellular effector that mediates phosphate control via a binding protein is thought to be a highly phosphorylated nucleotide that acts as a sensor of the phosphate level in the culture medium.18 This type of regulation could explain the immediate decrease observed in vancomycin production when increasing the medium phosphate content in chemostat culture (Fig. 3).

Production of the antibiotics nikkomycin and juglomycin by Streptomyces tendae are tightly regulated by the phosphate concentration of the medium: high phosphate stimulates juglomycin and decreases nikkomycin production and the opposite occurs when the concentration of phosphate is lowered.38 In both phosphate and glucose limited chemostat culture, specific growth rate is another important factor regulating vancomycin production; qvancomycin increased as growth rate increased to 0.087 h-’ before decreasing at the highest growth rate tested. The imposition of phosphate limitation mini- mized the repressive effect of phosphate on production, and qvancomycin was threefold higher than that under glucose limitation.

Similar studies in chemostat culture have revealed 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 . ~ , ~ ~ , ~ ~ In these chemostat cultures biomass concentration declined dramatically at the highest growth rates tested, suggesting that A. orientalis was unable to sustain a constant growth efficiency and vancomycin biosynthesis.

Results from these chemostat experiments has high- lighted several important physiological factors regulating vancomycin biosynthesis by A. orientalis strain ATCC 19795 in a defined medium. Information gained from these studies is enabling the physiological conditions controlling vancomycin production to be further investigated in high cell density cultures established in chemostats employing biomass recycling. Results of these investigations will be reported in a later publi- cation.

J. J. M. gratefully acknowledges a research studentship from BBSRC and thanks M. H. Goldman for assistance in amino acid HPLC analysis.

References

1. Arret, B., Johnson, D., Kirshbaum, A. 1971. Outline of details for microbiological assays of antibiotics. J. Pharmacol. Sci. 6 0 1689- 1694.

2. Bhatnagar, R. K., Doull J. L., Vining, L. C. 1988. Role of the carbon source in regulating chloramphenicol production in Strep- tomyces venezuelae: Studies in batch and continuous cultures. Can. J. Microbiol. 3 4 1217-1223.

3 Boeck, L., Mertz, F., Wolter, R., Higgins, C. 1984. N-DemethyI- vancomycin, a novel antibiotic produced by a strain of Nocardia orientalis. Taxonomy and fermentation. J. Antibiot. 37: 446- 453.

4. Bull, A. T., Huck, T. A., Bushell, M. E. 1990. Optimisation strate- gies in microbial process development and optimisation, pp. 145- 168. In: R. K. Poole, M. J. Bazin, and C. W. Keevil (eds.), Micro- bial growth dynamics. IRL Press, Oxford.

5. Castro, P. M., Hayter, P. M., Ison, A. P., Bull, A. T. 1992. Applica- tion of a statistical design to the optimisation of culture medium for recombinant interferon-gamma production by Chinese hamster ovary cells. Appl. Microbiol. Biotechnol. 38: 84-90.

6. Clark, G. J., Langley, D., Bushell, M. E. 1995. Oxygen limitation can induce microbial secondary metabolite formation: Investiga- tions with miniature electrodes in shaker and bioreactor culture. Microbiology 141 663-669.

7. Doull, J. F., Vining, L. C. 1990. Nutritional control of actinorh- rodin production by Streptomyces coelicolor A3(2): Suppression effects of nitrogen and phosphate. Appl. Microbiol. Biotechnol. 32: 449-454.

8. Fazeli, M. R., Cove, J. H., Baumberg, S. 1995. Physiological factors affecting streptomycin production by Streptomyces griseus ATCC 12475 in batch and continuous culture. FEMS Microbiol. Lett. U6: 55-62.

9. Garner, W. S . , Miller W. H. 1980. Reverse phase liquid chromatography analysis of amino acids after reaction with o-phthaldehyde. Anal. Biochem. 101: 61-65.

10, Goldberg, I., Er-El, Z. 1981. The chemostat-An efficient technique for medium optimisation. Proc. Biochem. 16: 2-8.

11. Hobbs, G., Fraser, C. M., Garner, D. C., Flett, F., Oliver, S. G. 1990. Pigmented antibiotic production by Streptomyces coelicolor A3(2): Kinetics and the influence of nutrients. J. Gen. Microbiol. 136 2291-2296.

12. James, P. D., Edwards, C., Dawson, M. 1991. The effects of temperature, pH and growth rate on secondary matabolism in Strepto- rnyces thermoviolaceus grown in chemostat culture. J. Gen. Micro- biol. 137 1715-1720.

13. Jehl, F., Gallion, C. Montail, H. 1987. Determination of vancomycin in human serum by high pressure liquid chromatography. Antimicrob. Agents Chemother. 2 7 503-507.

14. Kirby, R. 1992. The isolation and characteristics of antibiotic biosynthesis genes. Biotechnol. Adv. 10 561-576.

15. Kason, A. C., Egli, T. 1993. Dynamics of microbial growth in the deceleration and stationary phase of batch culture, pp. 103- 120. In: S. Kjelleberg (ed), Starvation in bacteria. Plenum Press, New York.

16. Lancini, G. C. 1989. Fermentation and biosynthesis of glycopeptide antibiotics. Prog. Ind. Microbiol. 2 7 283-296.

17. Lechevalier, M. P., Prauser, H., Labeda, D. P., Raun, J. S. 1986. Two new genera of Actinomycetes: Amycolatu gen. nov. and Amycolutopsis gen. nov. Int. J. Syst. Bacteriol. 2 4 29-37.

18. Liras, P., Asturias, J., Martin J. F. 1990. Phosphate control sequences involved in transcriptional regulation of antibiotic biosynthesis. TIBTECH. 8: 184-188.

19. Maimung, H. L., Wa-shai, H., Sherman, D. 1993. Precursor flux control targeted chromosomal insertion of the lysine aminotrans- ferase gene in cephamycin biosynthesis. J. Bacteriol. 175 6916- 6924.

20. Martin, J. F., Demain, A. L. 1980. Control of antibiotic biosynthesis. Microbiol. Rev. 44: 230-251.

21. McCormick, M. H. 1956. Vancomycin, a new antibiotic. Antibiot. Ann. 5 6 606-611.

22. McDermott, J. F., Letherbridge, G., Bushell, M. E. 1993. Estima- tion of the kinetic constants and elucidation of trends, in growth and erythromycin production in batch and continuous culture. Enzyme Microb. Technol. 1 5 657-668.

23. Mertz, F. P., Doolin, L. E. 1973. The effects of inorganic phosphate on the biosynthesis of vancomycin. Can. J. Microbiol. 19: 263- 270.

24. Monoghan, R. L., Koupal, R. D. 1989. Use of the Plackett Burman technique in the discovery program for new natural products, pp. 25-31. In: A. L. Demain, J. C. Hunter-Cevera, and W. W. Rosmore (eds.), Novel microbial products for medicine and agri- culture. Topics in industrial microbiology, Society for Industrial Microbiology, Elsevier, New York.

25. Murphy, J., Riley, J. P. 1962. A modified single solution method for the detection of phosphate in natural waters. Anal. Biochem. 2 7 31-36.

26. Nagaranjan, R. 1991. Antibacterial activity and mode of action of vancomycin and related glycopeptides. Antimicrob. Agents Chemother. 22: 605-609.

27. Noack, D. 1986. Directed selection of differential mutants of Strep- tomyces noursei using chemostat cultivation. J. Basic Microbiol.

28. Ozergin-Ulgen, K., Mavituna, F. 1994. Actinorhodin production by Sfreptomyces coelicolor A3(2): Kinetic parameter related to growth, substrate uptake and production. Appl. Microbiol. Bio- technol. 40: 457-462.

29. Pipersberg, W. 1994. Pathway engineering in secondary metabolite producing Actinomycetes. Crit. Rev. Biotechnob 1 4 252-285.

30. Pirt, S . J. 1975. Principles of microbe and cell cultivation, 2nd edition. Blackwell Scientific Publications, London.

31. Plackett, R. L., Burman, J. P. 1946. The design of multifactorial experiments. Biometrika 33: 305-325.

32. Rhodes, P. M. 1984. The production of oxytetracycline in chemostat culture. Biotechnol. Bioeng. 2 5 382-385.

33. Robinson, G. K., Alston, M. J., Knowles, C . J., Cheetham, P. S. J., Motion, K. R. 1994. An investigation into the factors influencing lipase-catalysed intramolecular lactonisation in microaqueous sys- tems. Enzyme Microb. Technol. 16: 855-863.

34. Roth, M., Noack, D. 1982. Genetic stability of differential func-

26: 231-239.


tions in Streptomyces hygroscopicus in relation to conditions of continuous culture. J. Gen. Microbiol. 128: 107-114.

35. Schuz, T., Fiedler, H., Zahner, H. 1993. Optimised nikkomycin production by fed-batch and continuous fermentation. Appl. Mi- crobiol. Biotechnol. 39: 433-437.

36. Strobel, R. J., Nakatsukasa, W. 1993. Response surface methods for optimising a novel macrolide producer. J. Ind. Microbiol.

37. Tang, L., Zhang, Y., Hutchinson, C . R. 1994. Amino acid catabo- lism and antibiotic synthesis: Valine a source of precursors for macrolide biosynthesis in Streptomyces ambofaciens and Strepto- myces fradiae. J. Bacteriol. 176: 6107-6119.

11: 121-127.

38. Treskatis, D. H., King, R., Wolf, H., Gilles, E. D. 1992. Nutritional control of nikkomycin and juglomycin production by Streptomyces tendae in continuous culture. Appl. Microbiol. Biotechnol. 36: 440-445.

39. Wasserman, G. F., Sitrin, R., Chapin, R. 1987. Affinitychromatog- raphy of the glycopeptide antibiotics. .I. Chromatogr. 392 225-238.

40. Whitaker, A. 1992. Actinomycetes in submerged culture. Appl. Biochem. Biotechnol. 32: 23-35.

41. Zarlenga, L. J., Gilmore, M. S., Sahm, D. F. 1992. Effects of amino acids on expression of enterococcal vancomycin resistance. Anti- microb. Agents Chemother. 36: 902-905.


Business

110315 vancomycin