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Comparison of the secondary metabolites in Penicillium chrysogenum between pilot and industrial penicillin G fermentations

  • Applied Microbial and Cell Physiology
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Abstract

The disparity of secondary metabolites in Penicillium chrysogenum between two scales of penicillin G fermentation (50 L as pilot process and 150,000 L as industrial one) was investigated by ion-pair reversed-phase liquid chromatography tandemed with hybrid quadrupole time-of-flight mass spectrometry. In industrial process, the pools of intracellular L-α-aminoadipyl-L-cysteinyl-D-valine (LLD-ACV) and isopenicillin N (IPN) were remarkably less than that in the pilot one, which indicated that the productivity of penicillin G might be higher in the large scale of fermentation. This conclusion was supported by the higher intracellular penicillin G concentration as well as its higher yield per unit biomass in industrial cultivation. The different changing tendencies of IPN, 6-aminopenicillanic acid and 6-oxopiperide-2-carboxylic acid between two processes also suggested the same conclusion. The higher content of intracellular LLD-ACV in pilot process lead to a similarly higher concentration of bis-δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine, which had an inhibitory effect on ACV synthetase and also subdued the activity of IPN synthetase. The interconversion of secondary metabolites and the influence they put on enzymes would intensify the discrepancy between two fermentations more largely. These findings provided new insight into the changes and regulation of secondary metabolites in P. chrysogenum under different fermentation sizes.

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References

  • Álvarez E, Cantoral JM, Barredo JL, Díez B, Martín JF (1987) Purification to homogeneity and characterization of the acyl-CoA: 6-APA acyltransferase of Penicillium chrysogenum. Antimicrob Agents Chemother 31:1675–1682

    Google Scholar 

  • Álvarez E, Meesschaert B, Montenegro E, Gutiérrez S, Díez B, Barredo JL, Martín JF (1993) The isopenicillin N acyltransferase of Penicillium chrysogenum has isopenicillin N aminohydrolase, 6-aminopenicillanic acid acyltransferase and penicillin amidase activities, all of which are encoded by the single penDE gene. Eur J Biochem 215:323–332

    Article  Google Scholar 

  • Brundidge SP, Gaeta FCA, Hook DJ, Sapino C, Elander RP, Morin RB (1980) Association of 6-oxo-piperidine-2-carboxylic acid with penicillin V production in Penicillium chrysogenum fermentations. J Antibiot 23:1348–1351

    Google Scholar 

  • Byford MF, Baldwin JE, Shiau CY, Schofield CJ (1997) The mechanisms of ACV synthetase. Chem Rev 97:2631–2650

    Article  Google Scholar 

  • Christensen L, Nielsen J, Villadsen J (1994) Degradation of penicillin V in fermentation media. Biotechnol Bioeng 44:165–169

    Article  CAS  Google Scholar 

  • Cohen G, Argaman A, Schreiber R, Mislovati M, Aharonowitz Y (1994) The thioredoxin system of Penicillium chrysogenum and its possible role in penicillin biosynthesis. J Bacteriol 176:973–984

    CAS  Google Scholar 

  • García-Estrada C, Vaca I, Lamas-Maceiras M, Martín JF (2007) In vivo transport of the intermediates of the penicillin biosynthetic pathway in tailored strains of Penicillium chrysogenum. Appl Microbiol Biotechnol 76:169–182.

    Article  Google Scholar 

  • Harris DM, Diderich JA, van der Krogt ZA, Luttik MA, Raamsdonk LM, Bovenberg RA, van Gulik WM, van Dijken JP, Pronk JT (2006) Enzymic analysis of NADPH metabolism in beta-lactam-producing Penicillium chrysogenum presence of a mitochondrial NADPH dehydrogenase. Metab Eng 8:91–101

    Article  CAS  Google Scholar 

  • Harvey LM, McNeil B, Berry DR, White S (1998) Autolysis in batch cultures of Penicillium chrysogenum at varying agitation rates. Enzyme Microb Technol 22:446–458

    Article  CAS  Google Scholar 

  • Henriksen CM, Holm SS, Schipper D, Jørgensen HS, Nielsen J, Villadsen J (1997a) Kinetic studies on the carboxylation of 6-amino-penicillanic acid to 8-hydroxy-penillic acid. Process Biochem 32:85–91

    Article  CAS  Google Scholar 

  • Henriksen CM, Nielsen J, Villadsen J (1997b) Influence of the dissolved oxygen concentration on the penicillin biosynthetic pathway in steady state cultures of Penicillium chrysogenum. Biotechnol Prog 13:776–782

    Article  CAS  Google Scholar 

  • Henriksen CM, Nielsen J, Villadsen J (1998) High exogenous concentrations of phenoxyacetic acid are crucial for a high penicillin V productivity in Penicillium chrysogenum. Microbiology-UK 144:2001–2006

    Article  CAS  Google Scholar 

  • Hersbach GJM, van der Beck CP, van Dijck PWM (1984) The penicillins: properties, biosynthesis and fermentation. In: Vandamme EJ (ed) Biotechnology of industrial antibiotics. Marcel Dekker, New York, pp 45–140

    Google Scholar 

  • Hsieh SH, Huang HY, Lee S (2009) Determination of eight penicillin antibiotics in pharmaceuticals, milk and porcine tissues by nano-liquid chromatography. J Chromatogr A 1216:7186–7194

    Article  CAS  Google Scholar 

  • Jaklitsch WM, Hampel W, Rohr M, Kubicek CP (1986) α-Aminoadipate pool concentration and penicillin biosynthesis in strains of Penicillium chrysogenum. Can J Microbiol 32:473–480

    Article  CAS  Google Scholar 

  • Jørgensen HS (1993) Metabolic fluxes in Penicillium chrysogenum. Ph.D. thesis, Technical University of Denmark, Lyngby, Denmark

  • Jørgensen HS, Nielsen J, Villadsen J, Mollgaard H (1995a) Analysis of penicillin V biosynthesis during fed-batch cultivations with a high yielding strain of Penicillium chrysogenum. Appl Microbiol Biotechnol 43:123–130

    Article  Google Scholar 

  • Jørgensen HS, Nielsen J, Villadsen J, Møllgaard H (1995b) Metabolic flux distributions in Penicillium chrysogenum during fed-batch cultivations. Biotechnol Bioeng 46:117–131

    Article  Google Scholar 

  • Junker BH, Hesse M, Burgess B, Masurekar P, Connors N, Seeley A (2004) Early phase process scale-up challenges for fungal and filamentous bacterial cultures. Appl Biochem Biotechnol 119:241–277

    Article  CAS  Google Scholar 

  • Jüsten P, Paul GC, Nienow AW, Thomas CR (1996) Dependence of mycelial morphology on impeller type and agitation intensity. Biotechnol Bioeng 52:634–648

    Article  Google Scholar 

  • Jüsten P, Paul GC, Nienow AW, Thomas CR (1998) Dependence of Penicillium chrysogenum growth, morphology, vacuolation, and productivity in fed-batch fermentations on impeller type and agitation intensity. Biotechnol Bioeng 59:762–775

    Article  Google Scholar 

  • Kleijn RJ, Liu F, van Winden WA, van Gulik WM, Ras C, Heijnen JJ (2007) Cytosolic NADPH metabolism in penicillin-G producing and non-producing chemostat cultures of Penicillium chrysogenum. Metab Eng 9:112–123

    Article  CAS  Google Scholar 

  • Lamas-Maceiras M, Vaca I, Rodriguez E, Casqueiro J, Martín JF (2006) Amplification and disruption of the phenylacetyl-CoA ligase gene of Penicillium chrysogenum encoding an aryl-capping enzyme that supplies phenylacetic acid to the isopenicillin N-acyltransferase. Biochem J 395:147–155

    Article  CAS  Google Scholar 

  • Larsson G, Enfors SO (1985) Influence of oxygen starvation on the respiratory capacity of Penicillium chrysogenum. Appl Microbiol Biotechnol 21:228–233

    Article  CAS  Google Scholar 

  • Lu WY, Kimball E, Rabinowitz JD (2006) A high-performance liquid chromatography-tandem mass spectrometry method for quantitation of nitrogen-containing intracellular metabolites. J Am Soc Mass Spectrom 17:37–50

    Article  CAS  Google Scholar 

  • Makagiansar HY, Ayazi Shamlou P, Thomas CR, Lilly MD (1993) The influence of mechanical forces on the morphology and penicillin production of Penicillium chrysogenum. Bioprocess Eng 9:83–90

    Article  CAS  Google Scholar 

  • Martin JF (2000) Alpha-aminoadipyl-cysteinyl-valine synthetases in beta-lactam producing organisms—from Abraham’s discoveries to novel concepts of non-ribosomal peptide synthesis. J Antibiot 53:1008–1021

    CAS  Google Scholar 

  • Mavrovouniotis ML (1990) Group contributions for estimating standard Gibbs energies of formation of biochemical compounds in aqueous solution. Biotechnol Bioeng 36:1070–1082

    Article  CAS  Google Scholar 

  • McNeil B, Berry DR, Harvey LM, Grant A, White S (1998) Measurement of autolysis in submerged batch cultures of Penicillium chrysogenum. Biotechnol Bioeng 57:297–305

    Article  CAS  Google Scholar 

  • Mu JM, Yi GC, Zhang JJ, Pang GR, Wang SL, Jiang L (2008) The study on inoculation amount of Penicillium chrysogenum during penicillin fermentation. Hebei Chem Eng 31:27–28

    Google Scholar 

  • Müller WH, van der Krift TP, Krouwer AJ, Woösten HA, van der Voort LH, Smaal EB, Verkleij AJ (1991) Localization of the pathway of the penicillin biosynthesis in Penicillium chrysogenum. EMBO J 10:489–495

    Google Scholar 

  • Müller WH, Bovenberg RA, Groothuis MH, Kattevilder F, Smaal EB, Van der Voort LH, Verkleij AJ (1992) Involvement of microbodies in penicillin biosynthesis. Biochim Biophys Acta 1116:210–213

    Google Scholar 

  • Müller WH, Essers J, Humbel BM, Verkleij AJ (1995) Enrichment of Penicillium chrysogenum microbodies by isopycnic centrifugation in nycodenz as visualized with immuno-electron microscopy. Biochim Biophys Acta 1245:215–220

    Google Scholar 

  • Nasution U, van Gulik WM, van Winden KRJ, WA PA, Heijnen JJ (2006) Measurement of intracellular metabolites of primary metabolism and adenine nucleotides in chemostat cultivated Penicillium chrysogenum. Biotechnol Bioeng 94:159–166

    Article  CAS  Google Scholar 

  • Nasution U, van Gulik WM, Ras C, Proell A, Heijnen JJ (2008) A metabolome study of the steady-state relation between central metabolism, amino acid biosynthesis and penicillin production in Penicillium chrysogenum. Metab Eng 10(1):10–23

    Article  CAS  Google Scholar 

  • Nielsen J (1997) Physiological engineering aspects of Penicillium chrysogenum. World Scientific, Singapore

    Google Scholar 

  • Nielsen J, Jørgensen HS (1995) Metabolic control analysis of the penicillin biosynthesis pathway in a high-yielding strain of P. chrysogenum. Biotechnology 11:299–305

    CAS  Google Scholar 

  • Nielsen J, Krabben P (1995) Hyphal growth and fragmentation of Penicillium chrysogenum in submerged cultures. Biotechnol Bioeng 46:588–598

    Article  CAS  Google Scholar 

  • Pang QL, Li QG (2006) The influence of corn liquid on industrial penicillin fermentation. Chinese Journal of Pharmaceuticals 37:528–530

    CAS  Google Scholar 

  • Paul GC, Thomas CR (1996) A structured model for hyphal differentiation and penicillin production using Penicillium chrysogenum. Biotechnol Bioeng 51:558–572

    Article  CAS  Google Scholar 

  • Paul GC, Kent CA, Thomas CR (1994) Hyphal vacuolation and fragmentation in Penicillium chrysogenum. Biotechnol Bioeng 44:655–660

    Article  CAS  Google Scholar 

  • Perry D, Abraham EP, Baldwin JE (1988) Factors affecting the isopenicillin N synthetase reaction. Biochem J 255:345–351

    CAS  Google Scholar 

  • Pissara P, Nielsen J, Bazin MJ (1996) Pathway kinetics and metabolic control analysis of a high-yielding strain of Penicillium chrysogenum during fed-batch cultivations. Biotechnol Bioeng 51:168–176

    Article  Google Scholar 

  • Ramos FR, López-Nieto MJ, Martín JF (1985) Isopenicillin N synthetase of Penicillium chrysogenum, an enzyme that converts delta-(l-alpha-aminoadipyl)-l-cysteinyl-d-valine to isopenicillin N. Antimicrob Agents Chemother 27:380–387

    CAS  Google Scholar 

  • Seifar RM, Zhao Z, van Dam J, van Winden W, van Gulik W, Heijnen JJ (2008) Quantitative analysis of metabolites in complex biological samples using ion-pair reversed-phase liquid chromatography-isotope dilution tandem mass spectrometry. J Chromatogr A 1187:103–110

    Article  CAS  Google Scholar 

  • Smith JJ, Lilly MD, Fox RI (1990) The effect of agitation on the morphology and penicillin production of Penicillium chrysogenum. Biotechnol Bioeng 35:1011–1023

    Article  CAS  Google Scholar 

  • Theilgaard HA, Nielsen J (1999) Metabolic control analysis of the penicillin biosynthetic pathway: the influence of the LLD-ACV:bisACV ratio on the flux control. Antonie van Leeuwenhoek J 75:145–154

    Article  CAS  Google Scholar 

  • Theilgaard HB, Kristiansen KN, Henriksen CM, Nielsen J (1997) Purification and characterization of δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine synthetase from Penicillium chrysogenum. Biochem J 327:185–191

    CAS  Google Scholar 

  • van Gulik WM, de Laat WT, Vinke JL, Heijnen JJ (2000) Application of metabolic flux analysis for the identification of metabolic bottlenecks in the biosynthesis of penicillin-G. Biotechnol Bioeng 68:602–618

    Article  Google Scholar 

  • van Gulik WM, Antoniewicz MR, deLaat WT, Vinke JL, Heijnen JJ (2001) Energetics of growth and penicillin production in a high-producing strain of Penicillium chrysogenum. Biotechnol Bioeng 72:185–193

    Article  Google Scholar 

  • van Holthoon F, Mulder PP, van Bennekom EO, Heskamp H, Zuidema T, van Rhijn HJ (2010) Quantitative analysis of penicillins in porcine tissues, milk and animal feed using derivatisation with piperidine and stable isotope dilution liquid chromatography tandem mass spectrometry. Anal Bioanal Chem 396:3027–3040

    Article  Google Scholar 

  • van Winden WA, van Gulik WM, Schipper D, Verheijen PJ, Krabben P, Vinke JL, Heijnen JJ (2003) Metabolic flux and metabolic network analysis of Penicillium chrysogenum using 2D [13C, 1H] COSY NMR measurements and cumulative bondomer simulation. Biotechnol Bioeng 83:75–92

    Article  Google Scholar 

  • Vardar F, Lilly MD (1982) Effect of cycling dissolved oxygen concentrations on product formation in penicillin fermentations. Eur J Appl Microbiol Biotechnol 14:203–211

    Article  CAS  Google Scholar 

  • Yang H, Allen DG (1999) Model-based scale-up strategy for mycelial fermentation processes. Can J Chem Eng 77:844–854

    Article  CAS  Google Scholar 

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Acknowledgment

The authors are grateful for the financial support from the National Natural Science Foundation of China (key program grant no. 20736006), the National Basic Research Program of China (“973” Program no. 2007CB714301), international collaboration project of MOST (2006DFA62400), Key Projects in the National Science & Technology Pillar Program (no. 2007BAD42B02), and Innovation Fund of Tianjin University.

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  1. Key Laboratory of Systems Bioengineering, Ministry of Education and Department of Pharmaceutical Engineering, School of Chemical Engineering and Technology, Tianjin University, P.O. Box 6888, Tianjin, 300072, People’s Republic of China

    Ying-Xiu Cao, Bin Qiao & Ying-Jin Yuan

  2. Hebei Zhongrun Pharmaceutical Co., Ltd, China, Shijiazhuang Pharmaceutical Group Co., Ltd. (CSPC), Shijiazhuang, Hebei Province, 050041, People’s Republic of China

    Hua Lu & Yao Chen

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  1. Ying-Xiu Cao
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  2. Bin Qiao
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  3. Hua Lu
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  5. Ying-Jin Yuan
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Correspondence to Ying-Jin Yuan.

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Cao, YX., Qiao, B., Lu, H. et al. Comparison of the secondary metabolites in Penicillium chrysogenum between pilot and industrial penicillin G fermentations. Appl Microbiol Biotechnol 89, 1193–1202 (2011). https://doi.org/10.1007/s00253-010-2910-y

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