Effect of glucose and oxygen on β-lactam biosynthesis by Cephalosporium acremonium

Journal of Biotechnology, 7 (1988) 131-140

131

Elsevier JBT 00292

Effect of glucose and oxygen on fl-lactam biosynthesis by Cephalosporium acremonium Alfred Scheidegger 1, Martin T. Kiienzi, Armin Fiechter 2 and Jakob Niiesch I International Research Laboratories, CIBA-GEIGY (Japan) Ltd, 10-66, Miyuki-cho, Takarazuka 665, Japan, Research Laboratories of the Pharmaceutical Dioision of CIBA-GEIGY Lta~ CH-4002 Basel, Switzerland and 2 Institute for Biotechnology, ETH-H5nggerberg~ CH-8093 Ziirich, Switzerland

(Received19 August1987; accepted13 November1987)

Summary The effects of glucose consumption rate (qs) and oxygen limitation on the control of cephalosporin C (Ceph C) biosynthesis and the activities of deacetoxycephalosporin C synthetase/hydroxylase (DAOC-SH) and acetyl coenzyme A:deacetylcephalosporin C o-acetyltransferase (DAC-AT) were investigated in cultivations of the highly productive Cephalosporium a c r e m o n i u m strain TR87 under conditions similar to those used in industrial production. A carefully optimised time course of qs during the first part of fed batch cultivations was essential for maximal Ceph C production. The actual glucose concentration in the medium was of secondary importance. A decrease of qs between 20 and 35 h of cultivation was found to induce the early onset of antibiotic synthesis. By subsequently maintaining qs at a relatively low level using a controlled feed of glucose and a limiting amount of phosphate, maximal production rates were obtained. Oxygen starvation after the onset of Ceph C production led to a pronounced increase in penicillin N formation, a reduced Ceph C yield ( - 30%) and a strongly reduced activity of the two enzymes tested. In general, neither the time course nor the absolute levels of the two enzyme activities directly correlated with the actual production rates of Ceph C. This is the first time where an independent parameter (qs) has been demonstrated to be responsible for triggering the synthesis of an antibiotic. Cephalosporium

acremonium;

Glucose regulation; Oxygen; Biosynthesis; Cepha-

losporin C; Enzyme Correspondence to: A. Scheidegger, International Research Laboratories, CIBA-GEIGY (Japan) Ltd,

10-66, Miyuki-cho,Takarazuka665, Japan. 0168-1656/88/$03.50 © 1988 ElsevierSciencePublishers B.V.(BiomedicalDivision)

132 Introduction

The development of a high yield Ceph C production process requires a good understanding of the main regulatory mechanisms governing growth and product formation. Glucose, inorganic phosphate and oxygen have long been recognized as important parameters in Ceph C synthesis. Furthermore, the use of a rapidly metabolisable carbon source such as glucose, can under certain conditions, lead to increased growth rates and reduced antibiotic formation (Kennel and Demain, 1978: Matsumura et al., 1978). In a purely synthetic medium containing excess phosphate, Ceph C production starts effectively after glucose is exhausted (Heim et al., 1984). The negative effect of glucose can be overcome by reducing phosphate supplementation to an appropriate level or by feeding growth limiting amounts of glucose (Kiienzi, 1980). Phosphate seems to exert its growth limiting effect indirectly by regulating the rate of glucose consumption. Measurements of the enzymes isopenicillin N synthetase and deacetoxycephalosporin C (DAOC-) synthetase revealed a strong glucose repression of the latter enzyme while the former was not affected (Behmer and Demain, 1983: Heim et al., 1984). This might explain why penicillin N (PenN) was produced in a glucose grown culture before the other fl-lactams appeared in the medium (Heim et al., 1984). In a high Ceph C producing strain higher levels of the two enzymes were measured than in a less efficient strain. The DAOC-synthetase appeared to be deregulated by carbon source repression (Shen et al., 1986). A more detailed analysis of the effect of glucose consumption on antibiotic production and enzyme levels has yet to be made. Many of the published results have been obtained with low producing strains and under conditions which are dissimilar to those used in industrial production. We were interested to see how a highly productive mutant strain of Cephalosporium acremonium is regulated by the supply of glucose and oxygen under well controlled conditions similar to those used in industrial practice. Furthermore, we wanted to see how the activities of enzymes involved in Ceph C biosynthesis were correlated with antibiotic production. Materials and Methods

Materials

a-Ketoglutarate was obtained from Serva, dithiothreitol (DTT) from Calbiochem, ATP disodium salt from Boehringer, Mannheim and phenylmethylsulfonyl fluoride (PMSF) from Sigma. PenN, DAOC, deacetylcephalosporin C and Ceph C were supplied by Ciba-Geigy Ltd. Organism, media and cultivation

The industrial, highly productive C. acremonium strain TR87 was grown under conditions described previously (Scheidegger et al., 1984). In addition to the

133 production medium with a low level of inorganic phosphate (P) (100 mg 1-1 P), a P-rich medium containing 5 g 1-1 KH2PO4 (800 mg 1-1 P) was used. The preparation of these solid-free media, containing mainly extracts of cornsteep, has been described earlier (Scheidegger et al., 1984). Glucose was fed to the production culture according to a program shown in Fig. 1. To study the effect of glucose, experiments were carried out with varied feed levels (absolute), while the basic feed pattern was maintained. After 54 h a soya oil feed at a rate of 0.9 g 1-1 h-1 was initiated in all batches. This feed rate was increased to 1.6 g 1-1 h-1 after 78 h and maintained until the end of cultivation.

Preparation of cell-free extracts Mycehum was obtained at different times by centrifugation and washed twice with cold deionized water. 10 g wet cells were suspended in 20 ml ice-cold buffer I (0.05 M Tris-HC1, pH 8.0, 0.01 M KC1, 0.01 M MgSO4, 0.01 M DTT, 0.001 M PMSF) and added to 20 g precooled glass beads (0.45-0.50 mm) in a 90 ml glass tube. Cells were ruptured by vibrating the mixture with an immersed glass piston attached to a Vibromixer (Chemap, Typ E 1) for 1 min (frequency 50 Hz). Cell debris were removed by centrifugation at 15 000 × g for 10 min. The proteins of the supernatant were then precipitated with 80% (NH4)2SO 4. The precipitate was collected by centrifugation and dissolved in buffer I. The solution was desalted on Sephadex G-25 and stored as extract A at - 6 0 o C.

Enzyme assays DA OC-SH The assay of DAOC-SH activities and the HPLC analysis of the products have been described elsewhere (Scheidegger et al., 1984). Extract A served as the enzyme solution (0.1-0.5 mg ml-1 protein, depending on enzyme activity). DAC-AT 1 ml of the standard reaction mixture contained 0.05 M Tris-HC1, pH 7.5, 4 mM MgSO4, 2 mM acetyl-CoA, 2 mM deacetylcephalosporin C (DAC) and extract A (0.05-0.5 mg ml-1 protein, depending on enzyme activity). The reaction was started by addition of DAC and the mixture incubated at 25 o C. After 10-45 min, the time varying according to the enzyme activity, the reaction was stopped by adding ethanol (1:1). The precipitated proteins were removed by centrifugation and the supernatant analyzed by HPLC, using a Zorbax BN-NH 2 (Dupont) column (4 × 250 mm) and a solvent system as described earlier (Scheidegger et al., 1985). Ceph C served as standard. One unit of DAOC-SH or DAC-AT activity is defined as the amount of enzyme which produces 1 #g DAOC + DAC or Ceph C min -1. The specific activity is defined as units per mg protein.

134

Analytical methods The biomass concentration (Cx) was determined as mycelial dry weight (d.w.) after centrifugation of duplicate 10 ml samples of medium, washing with deionised water twice and drying at 105 °C for 40 h. The concentration of glucose (cs) was assayed enzymatically using a glucose oxidase electrode. Soluble phosphate was determined colorimetrically and is expressed as phosphorus (P) (Harrison and Storr, 1944). PenN in the medium was estimated by HPLC, using a Lichrosorb RP-8 column and a solvent system consisting of 5% methanol in 0.01 M (NHa)2HPO 4, at pH 6.1. UV-absorption was measured at 222 nm. For Ceph C estimation (Cp), the same HPLC and solvent system were used but the pH of the solvent system was adjusted to 3.1. UV-absorption was measured at 262 nm. The partial oxygen tension ( p O 2 ) was measured on-line by a p O 2 electrode (Ingold, Switzerland). Protein was determined according to Bradford (1976). Bovine serum albumin was used as the standard.

Results

Optimal time course of the glucose uptake rate (qQ In a medium containing excess phosphate the availability of glucose and dependence on inoculum size and physiological state determine the process kinetics to a large extent. To obtain basic knowledge for the optimization of the Ceph C production process the effect of qs was investigated by different glucose supply regimes. The basic feed pattern is shown in Fig. 1 and was followed as closely as possible. Fig. 2 shows the course of a typical experiment with an absolute amount of fed glucose of 200 g 1-1 (measured after inoculation). The glucose concentration (cs) increased slightly in the first 25 h and then decreased to 0 (Fig. 2A). qs stayed at a mediocre level until about 60 h (Fig. 2B). When cx was at 40 g 1-1 the maximal Ceph C production rate (qp) was reached. A concentration of 14 g 1-1 Ceph C was obtained. In the ease of higher glucose supply (260 g 1-a) qs and qp passed through an earlier peak value (q~ exceeding 0.12 g g-1 h-1 at 20 h) and maximal qp was considerably lower resulting in a reduced final cp of only 11 g 1-1 Ceph C. A glucose supply lower than 200 g 1-1 led to an early exhaustion of glucose (at around 30 h) and a significant reduction of final %. These data indicate that the first 35 h of cultivation are decisive for the time of onset of Ceph C synthesis. The glucose supply must be sufficient to induce an early beginning of antibiotic production. Under such conditions qp can rise to its maximal value within the first 35 h even though glucose is still present in the medium. On the other hand, the experiments show clearly that too high q~ values in the early phase repress qp. However, limiting the glucose supply to qs less than 0.1 g

135

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Fig. 1. Glucose and soya oil feed after inoculation.The overall amount of glucose fed was varied without changing the character of the feed programme. g-1 h-1 during the first 25 h also has a strongly negative effect. The fragmentation of the mycelium is irreversibly impaired and as a result much lower Ceph C concentrations were obtained (data not shown). Thus, only a rapid decrease of qs after the initial period renders a stable production capacity resulting in a higher final cp. It can be concluded that optimal production would consist of a short initial period of relatively high glucose consumption followed by a rapid transition to a phase with a qs value less than 0.1 g g-1 h-1. In the experiments carried out no general correlation could be found between specific activities of DAOC-SH and DAC-AT and qp, qs or cp. Enzyme activities increase during the first 60 to 70 h to reach peak values but constantly decrease afterwards (Fig. 2C for the experiment described above). In order to initiate transition from high to low glucose consumption independent of the inoculum the content of phosphate in the medium was reduced. A reduction of inorganic phosphate from 800 to 100 mg 1-1 P was expected to limit qs even when a less active preculture was inoculated and a transient accumulation of glucose occurred. In the experiment illustrated in Fig. 3, a total of 185 g 1-1 glucose was used. At 30 h qs had decreased already from its early peak value to 0.08 g g-1 h-1 (Fig. 3B). After 60 h q~ was still at 0.072 g g-1 h-1. The production of Ceph C started as early as 30 h. qpmaX was considerably higher than in other experiments and was reached at 60 h when c X was already at 45 g 1-1. A final concentration of 19 g 1-1 Ceph C was obtained in 140 h. It is noteworthy that the activities of DAOC-SH and DAC-AT differ significantly from those in the earlier experiment. In this case the enzyme levels were generally higher and peak values of both enzymes were already found by 40 h (Fig. 3B). However, again there was no direct correlation of the specific activities with qp or q~.

136

Effect of oxygen starvation Growth as well as Ceph C biosynthesis by C. acremonium are oxygen-dependent processes• In vitro studies with D A O C - S H have shown that 0 2 is directly involved

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Fig. 2. Pattern of growth and production of Ceph C in the medium containing 800 mg l-1 p during cultivation of strain TR87. Total amount of fed glucose was 200 g 1-1. (A) Growth ( c , ) and concentration of Ceph C (Cp) and glucose (c,). (B) Specific glucose uptake rate (qs) and Ceph C productivity (qp). (C) Specific activity of DAOC-SH ( . . . . . . ) and DAC-AT (. . . . . . ).

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in the enzymatic reactions leading from PenN to DAC. The experiment shown in Fig. 4 was intended to show the effect of temporary 02 starvation on the synthesis of Ceph C and its precursor molecules. The experiment was carried out as described for the cultivation shown in Fig. 3 but the concentration of dissolved oxygen in the culture medium expressed as p 02 was allowed to drop below 10% saturation for about 12 h. Under standard conditions the stirring rate would have been increased during this period to avoid the dissolved oxygen level from falling below 20% saturation. This step was omitted. The lack of oxygen led to a strongly accelerated synthesis of PenN (Fig. 4A), which is below 1 g 1-1 under aerobic conditions. Simultaneously, a strong reduction of the activities of DAOC-SH and DAC-AT was observed (Fig. 4B). However, the

139 production of Ceph C and DAC initially was virtually unchanged. But in contrast to cultivations with no 02 starvation period Ceph C synthesis stopped earlier and the final concentration of Ceph C was reduced by 30%.

Discussion

The cultivation system used in this work has two main advantages. Due to the use of solid free media it is possible to determine specific consumption and production rates and at the same time maintain conditions which are similar to those used in industrial practice. Furthermore, the application of a programmed feed makes it possible to control the substrate consumption more directly in contrast to batch conditions. A shortcoming of the system is its limited ability to compensate for variations in the physiological state of the precultures. The system could be improved by regulating the glucose feed according to the actual metabolic activity of a particular culture, but this would require sensitive on-line measurements of growth indicators. The results obtained confirm experience made with industrial Ceph C processes. The antibiotic production is strongly dependent on the consumption of the carbon source during the first growth phase. Too high qs values initially prevent the development of a high rate of Ceph C synthesis. As already shown in a purely synthetic medium (Kiienzi, 1980) it is not the concentration of glucose but its rate of consumption which regulates antibiotic production. Prerequisites for an early onset and a subsequent sustained high level of Ceph C production are an early transition of the culture from a short period of relatively high qs to a period of reduced q~. This goal was reached by applying an appropriate feed regime in combination with a reduced phosphate level in the medium. In industrial practice supplementation of a slowly metabolisable polysaccharide as growth limiting carbon source for the first phase is preferred. By using soya oil as a carbon source during the second phase of the process DAC production and foaming could be reduced. According to the results of Heim et al., (1984) and Shen et al. (1986) the sensitivity of DAOC-synthetase to glucose repression is strain dependent. Since the Ceph C synthesis by TR87 was found to be repressed by glucose it was expected that this regulatory effect would be reflected at the enzyme level. However, the results show that there is no straight-forward relationship between qs and qp, on the one hand, and the specific enzyme activities, on the other. In general, experiments with high final Ceph C concentrations did also have higher levels of DAOC-SH and DAC-AT. However, no correlation seems to exist between the time course of qp and the enzyme activities. Furthermore, the culture grown under oxygen limitation did produce a high amount of Ceph C although the DAOC-SH and DAC-AT activities were very low. The increased formation of PenN under these conditions can most probably be attributed to an intracellular deficit of oxygen which is a cofactor for the DAOC-SH. It seems unlikely that for strain TR87 the amount of DAOC-SH and DAC-AT play a major role in regulating Ceph C synthesis. Since isopenicillin N

140

synthetase, isopenicillin N isomerase and DAOC-synthetase were shown to be regulated in a concerted way (Ramos et al., 1986), enzymes which catalyse earlier steps in the biosynthesis of fl-lactams must be considered more likely as candidates for glucose repression.

Acknowledgements The authors thank J.A.L. Auden for reviewing the manuscript and A. Gutzwiller for his experimental assistance.

References Behmer, C.J. and Demain, A.L. (1983) Further studies on carbon catabolite regulation of fl-lactam antibiotic synthesis in Cephalosporium acremonium. Curr. Microbiol. 8, 107-114. Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254. Harrison, T.S. and Storr, H. (1944) The determination of soluble phosphate and silica in water by means of the Spekker photometric absorptiometer. J. Soe. Chem. Ind. 63, 154-157. Heim, J., Shen, Y.-Q., Wolfe, S. and Demain, A.L. (1984) Regulation of isopenicillin N synthetase and deacetoxycephalosporin C synthetase by carbon source during the fermentation of Cephalosporium acremonium. Appl. Microbiol. Biotechnol. 19, 232-236. Kennel, Y.M. and Demain, A.L. (1978) Effect of carbon sources on fl-lactam antibiotic formation by Cephalosporium acremonium. Exp. Mycol. 2, 234-238. Kiienzi, M.T. (1980) Regulation of cephalosporin synthesis in Cephalosporium acremonium by phosphate and glucose. Arch. Microbiol. 128. 78-83. Matsumura, M., Imanaka, T., Yoshida, T. and Taguchi, H. (1978) Effect of glucose and methionine consumption rates on cephalosporin C production by Cephalosporium acremonium. J. Ferment. Technol. 56, 345-353. Ramos, F.R., Lopez-Nieto, M.J. and Martin, J.F. (1986) Coordinate increase of isopenicillin N synthetase, isopenicillin N epimerase and deacetoxycephalosporin C synthetase in a high cephalosporinproducing mutant of Acremonium chrysogenum and simultaneous loss of the enzymes in a non-producing mutant. FEMS Microbiol. Lett. 35, 123-127. Scheidegger, A., Kiienzi, M.T. and Niiesch, J. (1984) Partial purification and catalytic properties of a bifunctional enzyme in the biosynthetic pathway of fl-lactams in Cephalosporium acremonium. J. Antibiot. 37. 522-531. Scheidegger, A., Gutzwiller, A., Kiienzi, M.T., Fiechter, A. and Niiesch, J. (1985) Investigation of acetyl-CoA: Deacetylcephalosporin C o-acetyltransferase of Cephalosporium acremonium. J. Biotechnol. 3, 109-117. Shen, Y.-Q., Wolfe, S. and Demain, A.L. (1986) Levels of isopenicillin N synthetase and deacetoxycephalosporin C synthetase in Cephalosporium acremonium producing high and low levels of cephalosporin C. Bio/Technology 4, 61-64.