ПРИКЛАДНАЯ БИОХИМИЯ И МИКРОБИОЛОГИЯ, 2013, том 49, № 2, с. 190-196
UDC 576.852.1
BIOCHEMICAL PARAMETERS OF Saccharopolyspora erythraea DURING FEEDING AMMONIUM SULPHATE IN ERYTHROMYCIN BIOSYNTHESIS
PHASE
© 2013 X. Zou*, **, W.-J. Li***, W. Zeng***, H.-F. Hang*, J. Chu*, Y.-P. Zhuang*, S.-L. Zhang**
* East China University of Science and Technology, Shanghai 200237, China ** Southwest University, Chongqing 400715, China ***Yidu HEC Biochem. Co. Ltd, Hubei 443300, China e-mail: juchu@ecust.edu.cn Received February 8, 2012
The physiology of feeding ammonium sulphate in erythromycin biosynthesis phase of Saccharopolyspora erythraea on the regulation of erythromycin A (Er-A) biosynthesis was investigated in 50 L fermenter. At an optimal feeding ammonium sulphate rate of 0.03 g/L per h, the maximal Er-A production was 8281 U/mL at 174 h of growth, which was increased by 26.3% in comparison with the control (6557 U/mL at 173 h). Changes in cell metabolic response of actinomycete were observed, i.e. there was a drastic increase in the level of carbon dioxide evolution rate and oxygen consumption. Assays of the key enzyme activities and organic acids of S. erythraea and amino acids in culture broth revealed that cell metabolism was enhanced by ammonium assimilation, which might depend on the glutamate transamination pathway. The enhancement of cell metabolism induced an increase of the pool of TCA cycle and the metabolic flux of erythromycin biosynthesis. In general, ammonium assimilation in the erythromycin biosynthesis phase of S. erythraea exerted a significant impact on the carbon metabolism and formation of precursors of the process for dramatic regulation of secondary metabolites biosynthesis.
DOI: 10.7868/S0555109913020189
Erythromycin is a polyketide antibiotic produced by Saccharopolyspora erythraea in submerged fermentation. Erythromycin A (Er-A) is the main active component of erythromycin products. Simultaneously, there exist two structurally similar by-products of eryhtromycin B (Er-B) and eryhtromycin C (Er-C) in the culture broth. In fermentation phase, improving Er-A production is an effective way to meet the market demands and reduce the production cost.
Ammonium salts are an available inorganic nitrogen source, which usually regarded to suppress the biosynthesis of antibiotics [1]. Addition of ammonium ions (5—30 mM) to Penicillium urticae shake-flask culture led to a strong repression of the enzyme activities of secondary metabolism [2]. However, in cepha-losporin-C fermentation, ammonium sulphate as inorganic nitrogen source was suitable for higher yield of antibiotic [3]. In previous work, we first reported that feeding ammonium sulphate in erythromycin biosynthesis phase of S. erythraea could affect Er-A biosynthesis [4]. Nevertheless, a thorough understanding of the physiology mechanism of ammonium assimilation for enhancing Er-A production was still lacking.
As we know, there are close links between primary and secondary metabolism, both in terms of precursors formation and nutrient regulation. Er-A has 3
structural parts with the 14-member macrolide ring and 2 deoxysugars, in which the lactonic ring requires propionyl-CoA and 6 methylmalonyl-CoA molecules as precursors for its biosynthesis [5]. These compounds could have multiple metabolic origin including catabolism of odd-numbered fatty acids, reduction of acrylate, rearrangement of succinyl-coenzyme A and catabolism of Met, Thr or Val [6, 7]. Deeper knowledge of intermediary metabolism, especially of TCA cycle enzymes and precursors supply, may provide a better understanding of the link between primary and secondary metabolism for Er-A biosynthesis.
The aim of the study was to investigate some key enzymes and metabolites profiles in carbon metabolism, and amino acids utilization in culture broth of S. erythraea under the optimal feeding ammonium sulphate rate in erythromycin biosynthesis phase using 50 L fermenter. The results showed that cell metabolism was enhanced by ammonium assimilation, which induced an increase of the pool of TCA cycle and the metabolic flux of erythromycin biosynthesis. The information obtained in this work should be helpful for deeper understanding of ammonium assimilation in erythromycin biosynthesis phase for the regulation of Er-A biosynthesis in S. erythraea.
MATERIALS AND METHODS
Microorganism and culture conditions. Saccharopolyspora erythraea № 8, an erythromycin producer strain from Yidu HEC Biochem. Co. Ltd. (Hubei, China) was used, which was publicly available [9]. Agar slants were inoculated with spores and incubated at 32°C for 7 days, and then used for seed culture inoculation. For seed cultures, the medium composition was (g/L): starch — 30, soybean flour — 15, NaCl — 5, (NH4)2SO4 — 2. The seed culture was grown in a 500 mL shake flask containing 50 mL ofliquid medium and incubated at 32°C on a rotary shaker (220 rpm) for 7 days. The fermentation cultivation was inoculated with 10% (v/v) of the above seed culture medium and incubated at 33°C.
Feeding ammonium sulphate in erythromycin biosynthesis phase. The 50 L fermenter was manufactured by Shanghai Guoqiang Bioengineering Equipment Co., Ltd. (China) [8], which had a 30 L (working volume) agitated bioreactor with 3 turbine impellers and equipped with devices to monitor and control more than 14 on-line measurable parameters. The stirred reactor was aerated through a ring sparger. Dissolved oxygen (DO) level was set above 20% of air saturation and controlled by adjusting agitation speed and aeration rate during fermentation. DO concentration was detected using polarographic DO electrode (Mettler Toledo, Switzerland). The CO2 off-gas from the fermenter was measured with the gas analyzer (Chongqing Hateman measuring instruments Co., Ltd, China). The cultivation temperature was kept at 33°C, and pH was controlled at 6.9—7.0 with feeding glucose concentration of 300 g/L during the whole of fermentation process. Three independent samples were taken every 8 h for the analyses of cell biomass, erythromycin
production, NH+ and total sugar concentration.
Feeding ammonium sulphate was commenced from 80 h to the end of fermentation in 50 L fermenter. The rate of the process was 0.03 g/L per h. Cultivation without feeding ammonium sulphate was used as the control.
Determination of cell biomass (packed mycelium volume, PMV). For the determination of cell biomass (PMV), 10 ml of culture broth was taken as sample, after removal of supernatant by centrifugation (4,000 x g, 10 min), PMV was calculated as the volume of precipitate/10 mL of culture broth.
Analysis of extra- and intracellular organic acids.
For the determination of extra- and intracellular organic acids of metabolism, the HPLC system (Aligent 1200, USA) was equipped with AquaSep C8 column and a UV detector (210 nm). 0.6 mL/min mobile phase using 0.01mol/L H3PO4 solution was applied to the column. The column was operated at 30°C [9].
Analysis of amino acids. For the determination of free amino acids in culture broth, the amino acids were derived by automatic pre-column O-phthaldialdehyde (OPA) derivation methods. The HPLC was equipped with Agilent 1200 system, column ZORBAX Eclipse-AAA (4.6 mm x 150 mm, 5 ^m, Agilent, USA). Mobile phase contained mixture of 0.04 M NaH2PO4, pH 7.0, as A phase and acetonitrile, methanol and distilled water (45 : 45 : 5, v/v) as B phase. Flow rate was 2 mL/min using UV detector at 238 nm.
Analysis of enzyme activities in vitro. Preparation of cell extracts. Enzyme activity was defined as ^mol of substrate consumed per min and mg of protein (U/mg). In each case, reactor bulk liquid samples were withdrawn and centrifuged at 12,000 x g and 4°C for 15 min. The supernatant was removed and cells were resuspended with 50 mM potassium phosphate extraction buffer (pH 7.4), the ratio of buffer : cells was 10 : 1. Cells were lysed with addition of lysozyme (1 g/mL of buffer) at room temperature for 2 h. The extract was centrifuged at 12,000 x g and 4°C for 15 min to remove cell debris and the supernatant was used for subsequent activity measurements. Protein content was determined by Bradford assay method.
Pyruvate kinase. The activity of pyruvate kinase (PKS) was measured spectrophotometrically [10]. All compounds of the reaction mixture were pipetted into a colorimetric ware, and reactions initiated by adding the cell extract to give a final volume of 1 mL. The reaction mixture contained 80 mM Tris-HCl buffer (pH 7.4), 10 mM MgCl2, 2 mM ADP, 10 mM phosphoenolpyru-vate, 2 mM NADH, and 5 U lactate dehydrogenase (Shanghai Yuanju Biotechnology Co., Ltd., China).
Citrate synthetase. The activity of citrate synthetase (CS) was detected by monitoring the disappearance of NADH during reaction [11]. The assay conditions were the following: 2 mM 5,5'-dithiobis-(2-nitroben-zoic acid), 0.5 mM Tris-HCl buffer (pH 7.4), 0.2 mM acetyl-CoA, and 0.15 mM oxaloacetic acid.
Glutamine synthetase. The activity of glutamine synthetase (GS) was determined by measuring the increase of glutamylhydroxamate at 540 nm [12]. The reaction mixture contained 30 mM glutamate, 0.4 mM ADP, 20 mM sodium dihydrogen arsenate, 60 mM hy-droxylamine, 3 mM MnCl2 in 40 mM imidazole buffer (pH 7.0) and cell extract in a total volume of 1 mL.
Methylmalonyl coenzyme A mutase. Methylmalonyl coenzyme A mutase (MCM) was assayed as reported by Bermudez et al. [6] with minor modifications. The activity was detected by measuring the decreased suc-cinyl-CoA by HPLC. The reaction mixture contained 1 mM dithioerythritol, 60 ^M coenzyme B12, 50 ^M succinyl-CoA in 100 mM Tris-buffer (pH 8.0) and cell extract in a total volume of 1 mL. The reaction was initiated by addition of succinyl-CoA and incubated in
6 ПРИКЛАДНАЯ БИОХИМИЯ И МИКРОБИОЛОГИЯ том 49 № 2 2013
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Fig. 1. The changes of the CER and DO level during growth of S. erythrae
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