Skip to main content
SpringerLink
Log in

Optimization of phenazine-1-carboxylic acid production by a gacA/qscR-inactivated Pseudomonas sp. M18GQ harboring pME6032Phz using response surface methodology

  • Biotechnological Products and Process Engineering
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

Phenazine-1-carboxylic acid (PCA) production was enhanced in Pseudomonas sp. M18 wild strain and its mutants carrying recombinant pME6032Phz for phz gene cluster overexpression, among which Pseudomonas sp. strain M18GQ/pME6032Phz, a gacA and qscR double gene chromosomally inactivated mutant harboring pME6032Phz, showed the highest PCA yield. The conditions for fermentation and isopropyl-β-d-1-thiogalactopyranoside (IPTG) induction were optimized for strain M18GQ/pME6032Phz in shake flask experiments. A one-factor-at-a-time approach, followed by a fractional factorial design identified soybean meal, corn steep liquor, and ethanol as statistically significant factors. Optimal concentrations and mutual interactions of the factors were then determined by the method of steepest ascent and by response surface methodology based on the center composite design. The predicted PCA production was 6,335.2 mg/l after 60 h fermentation in the optimal medium of 65.02 g soybean meal, 15.36 g corn steep liquor, 12 g glucose, 21.70 ml ethanol, and 1 g MgSO4 per liter in the flask fermentations, with induction of 1.0 mmol/l IPTG 24 h after inoculation. In an experimental validation under these conditions, the maximum PCA production was 6,365.0 mg/l. This represents a ∼60% increase over production by strain M18GQ in optimal conditions. The negative effect of plasmid pME6032 on the expression of chromosomally located phz gene cluster was found in Pseudomonas sp. M18GQ, and the possible reason was discussed in the text.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  • Butler MJ, Takano E, Bruheim P, Jovetic S, Marinelli F, Bibb MJ (2003) Deletion of scbA enhances antibiotic production in Streptomyces lividans. Appl Microbiol Biotechnol 61:512–516

    CAS  Google Scholar 

  • Daly R, Hearn MT (2005) Expression of heterologous proteins in Pichia pastoris: a useful experimental tool in protein engineering and production. J Mol Recognit 18:119–138

    Article  CAS  Google Scholar 

  • Deepak V, Kalishwaralal K, Ramkumarpandian S, Babu SV, Senthilkumar SR, Sangiliyandi G (2008) Optimization of media composition for Nattokinase production by Bacillus subtilis using response surface methodology. Bioresour Technol 99:8170–8174

    Article  CAS  Google Scholar 

  • Donovan RS, Robinson CW, Glick BR (2000) Optimizing the expression of a monoclonal antibody fragment under the transcriptional control of the Escherichia coli lac promoter. Can J Microbiol 46:532–541

    Article  CAS  Google Scholar 

  • Ge Y, Huang X, Wang S, Zhang X, Xu Y (2004) Phenazine-1-carboxylic acid is negatively regulated and pyoluteorin positively regulated by gacA in Pseudomonas sp. M18. FEMS Microbiol Lett 237:41–47

    Article  CAS  Google Scholar 

  • Gibson J, Sood A, Hogan DA (2009) Pseudomonas aeruginosa-Candida albicans interactions: localization and fungal toxicity of a phenazine derivative. Appl Environ Microbiol 75:504–513

    Article  CAS  Google Scholar 

  • Graupner S, Wackernagel W (2000) A broad-host-range expression vector series including a Ptac test plasmid and its application in the expression of the dod gene of Serratia marcescens (coding for ribulose-5-phosphate 3-epimerase) in Pseudomonas stutzeri. Biomol Eng 17:11–16

    Article  CAS  Google Scholar 

  • Heeb S, Itoh Y, Nishijyo T, Schnider U, Keel C, Wade J, Walsh U, O’Gara F, Haas D (2000) Small, stable shuttle vectors based on the minimal pVS1 replicon for use in Gram-negative, plant-associated bacteria. Mol Plant Microb Interact 13:232–237

    Article  CAS  Google Scholar 

  • Hu HB, Xu YQ, Chen F, Zhang XH, Hur BK (2005) Isolation and characterization of a new fluorescent Pseudomonas strain that produces both phenazine-1-carboxylic acid and pyoluteorin. J Microbiol Biotechnol 15:86–90

    CAS  Google Scholar 

  • Huang J, Xu Y, Zhang H, Li Y, Huang X, Ren B, Zhang X (2009) Temperature-dependent expression of phzM with its regulatory genes lasI and ptsP in rhizosphere isolated Pseudomonas sp. Strain M18. Appl Environ Microbiol 75:6568–6580

    Article  CAS  Google Scholar 

  • Jiang K, Pi Y, Hou R, Zeng H, Huang Z, Zhang Z, Sun X, Tang K (2009) Molecular cloning and expression profiling of the first specific jasmonate biosynthetic pathway gene allene oxide synthase from Lonicera japonica. Mol Biol Rep 36:487–493

    Article  CAS  Google Scholar 

  • Kennedy MM, Krouse D (1999) Strategies for improving fermentation medium performance: a review. J Ind Microbiol Biotech 23:456–475

    Article  CAS  Google Scholar 

  • Khuri AI, Cornell JA (1987) Response surfaces: design and analysis. Marcel Decker Inc., New York

    Google Scholar 

  • Kumar RS, Ayyadurai N, Pandiaraja P, Reddy AV, Venkateswarlu Y, Prakash O, Sakthivel N (2005) Characterization of antifungal metabolite produced by a new strain Pseudomonas aeruginosa PUPa3 that exhibits broad-spectrum antifungal activity and biofertilizing traits. J Appl Microbiol 98:145–154

    Article  CAS  Google Scholar 

  • Li Y, Jiang H, Du X, Huang X, Xu Y (2010) Enhancement of phenazine-1-carboxylic acid production using batch and fed-batch culture of gacA inactivated Pseudomonas sp. M18G. Bioresour Technol (in press)

  • Li Y, Jiang H, Xu Y, Zhang X (2008) Optimization of nutrient components for enhanced phenazine-1-carboxylic acid production by gacA-inactivated Pseudomonas sp. M18G using response surface method. Appl Microbiol Biotechnol 77:1207–1217

    Article  CAS  Google Scholar 

  • Li Y, Lu J (2005) Characterization of the enzymatic degradation of arabinoxylans in grist containing wheat malt using response surface methodology. J Am Soc Brew Chem 63:171–176

    CAS  Google Scholar 

  • Mavrodi DV, Bonsall RF, Delaney SM, Soule MJ, Phillips G, Thomashow LS (2001) Functional analysis of genes for biosynthesis of pyocyanin and phenazine-1-carboxamide from Pseudomonas aeruginosa PAO1. J Bacteriol 21:6454–6465

    Article  Google Scholar 

  • Mazzola M, Cook RJ, Thomashow LS, Weller DM, Pierson LS 3rd (1992) Contribution of phenazine antibiotic biosynthesis to the ecological competence of fluorescent pseudomonads in soil habitats. Appl Environ Microbiol 58:2616–2624

    CAS  Google Scholar 

  • Mundra P, Desai K, Lele SS (2007) Application of response surface methodology to cell immobilization for the production of palatinose. Bioresour Technol 98:2892–2896

    Article  CAS  Google Scholar 

  • Muralidhar RV, Chirumamilla RR, Marchant R, Nigam P (2001) A response surface approach for the comparison of lipase production by Candida cylindracea using two different carbon sources. Biochem Eng J 9:17–23

    Article  CAS  Google Scholar 

  • Raaijmakers JM, Weller DM, Thomashow LS (1997) Frequency of antibiotic-producing Pseudomonas spp. in natural environments. Appl Environ Microbiol 63:881–887

    CAS  Google Scholar 

  • Reynard LN, Cocquet J, Burgoyne PS (2009) The multi-copy mouse gene Sycp3-like Y-linked (Sly) encodes an abundant spermatid protein that interacts with a histone acetyltransferase and an acrosomal protein. Biol Reprod 81:250–257

    Article  CAS  Google Scholar 

  • Sambrook J, Russell D (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor

    Google Scholar 

  • Sayyad SA, Panda BP, Javed S, Ali M (2007) Optimization of nutrient parameters for lovastatin production by Monascus purpureus MTCC 369 under submerged fermentation using response surface methodology. Appl Microbiol Biotechnol 73:1054–1058

    Article  CAS  Google Scholar 

  • Shtark O, Shaposhnikov AI, Kravchenko LV (2003) The production of antifungal metabolites by Pseudomonas chlororaphis grown on different nutrient sources. Mikrobiologiia 72:645–650

    Google Scholar 

  • Slininger PJ, Jackson MA (1992) Nutritional factors regulating growth and accumulation of phenazine-1-carboxylic acid by Pseudomonas fluorescens 2–79. Appl Microbiol Biotechnol 37:388–392

    Article  CAS  Google Scholar 

  • Stock R, Grant RJ, Klopfenstein TJ (1995) Average composition of feeds used in Nebraska. Institute of Agricultural Resources, University of Nebraska Lincoln, Lincoln

    Google Scholar 

  • Su J, Zhou Q, Zhang H, Li Y, Huang X, Xu Y (2009) Medium optimization for phenazine-1-carboxylic acid production by a chromosomally inactivated gacA and qscR double mutant Pseudomonas sp. M18GQ using response surface methodology. Bioresour Technol (in press)

  • Tang XJ, He GQ, Chen QH, Zhang XY, Ali MA (2004) Medium optimization for the production of thermal stable beta-glucanase by Bacillus subtilis ZJF-1A5 using response surface methodology. Bioresour Technol 93:175–181

    Article  CAS  Google Scholar 

  • Thomashow LS, Weller DM (1988) Role of a phenazine antibiotic from Pseudomonas fluorescens in biological control of Gaeumannomyces graminis var. tritici. J Bacteriol 170:3499–3508

    CAS  Google Scholar 

  • Thomashow LS, Weller DM, Bonsall RF, Pierson LS (1990) Production of the antibiotic phenazine-1-carboxylic acid by fluorescent Pseudomonas species in the rhizosphere of wheat. Appl Environ Microbiol 56:908–912

    CAS  Google Scholar 

  • Wang W, Wen X (2009) Expression of lignin peroxidase H2 from Phanerochaete chrysosporium by multi-copy recombinant Pichia strain. J Environ Sci (China) 21:218–222

    CAS  Google Scholar 

  • Wang Y, Huang X, Hu H, Zhang X, Xu Y (2008) QscR acts as an intermediate in gacA-dependent regulation of PCA biosynthesis in Pseudomonas sp. M-18. Curr Microbiol 56:339–345

    Article  CAS  Google Scholar 

  • Wlad H, Ballagi A, Bouakaz L, Gu Z, Janson JC (2001) Rapid two-step purification of a recombinant mouse Fab fragment expressed in Escherichia coli. Protein Expr Purif 22:325–329

    Article  CAS  Google Scholar 

  • Xiao ZJ, Liu PH, Qin JY, Xu P (2007) Statistical optimization of medium components for enhanced acetoin production from molasses and soybean meal hydrolysate. Appl Microbiol Biotechnol 74:61–68

    Article  CAS  Google Scholar 

  • Xiong ZQ, Tu XR, Tu GQ (2008) Optimization of medium composition for actinomycin X2 production by Streptomyces spp JAU4234 using response surface methodology. J Ind Microbiol Biotechnol 35:729–734

    Article  CAS  Google Scholar 

  • Yuan LL, Li YQ, Wang Y, Zhang XH, Xu YQ (2008) Optimization of critical medium components using response surface methodology for phenazine-1-carboxylic acid production by Pseudomonas sp. M-18Q. J Biosci Bioeng 105:232–237

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported in part by grants from the Major State Basic Research Development Program of China (973 Program; no. 2009CB118906), the National High Technology Research and Development Program of China (863 Program; nos. 2006AA10A209 and 2006AA02Z228), and the Shanghai Science and Technology Program (no. 08391911900).

Author information

Authors and Affiliations

  1. Key Laboratory of Microbial Metabolism, Ministry of Education, School of Life Science and Biotechnology, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai, 200240, People’s Republic of China

    Quan Zhou, Jinjiang Su, Haixiang Jiang, Xianqing Huang & Yuquan Xu

Authors
  1. Quan Zhou
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jinjiang Su
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Haixiang Jiang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Xianqing Huang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Yuquan Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Yuquan Xu.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Zhou, Q., Su, J., Jiang, H. et al. Optimization of phenazine-1-carboxylic acid production by a gacA/qscR-inactivated Pseudomonas sp. M18GQ harboring pME6032Phz using response surface methodology. Appl Microbiol Biotechnol 86, 1761–1773 (2010). https://doi.org/10.1007/s00253-010-2464-z

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-010-2464-z

Keywords

Search

Navigation