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Enhanced production of l-phenylalanine in Corynebacterium glutamicum due to the introduction of Escherichia coli wild-type gene aroH

  • Metabolic Engineering and Synthetic Biology
  • Published:
Journal of Industrial Microbiology & Biotechnology

Abstract

Metabolic engineering is a powerful tool which has been widely used for producing valuable products. For improving l-phenylalanine (l-Phe) accumulation in Corynebacterium glutamicum, we have investigated the target genes involved in the biosynthetic pathways. The genes involved in the biosynthesis of l-Phe were found to be strictly regulated genes by feedback inhibition. As a result, overexpression of the native wild-type genes aroF, aroG or pheA resulted in a slight increase of l-Phe. In contrast, overexpression of aroF wt or pheA fbr from E. coli significantly increased l-Phe production. Co-overexpression of aroF wt and pheA fbr improved the titer of l-Phe to 4.46 ± 0.06 g l−1. To further analyze the target enzymes in the aromatic amino acid synthesis pathway between C. glutamicum and E. coli, the wild-type gene aroH from E. coli was overexpressed and evaluated in C. glutamicum. As predicted, upregulation of the wild-type gene aroH resulted in a remarkable increase of l-Phe production. Co-overexpression of the mutated pheA fbr and the wild-type gene aroH resulted in the production of l-Phe up to 4.64 ± 0.09 g l−1. Based on these results we conclude that the wild-type gene aroH from E. coli is an appropriate target gene for pathway engineering in C. glutamicum for the production of aromatic amino acids.

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References

  1. Baez-Viveros JL, Osuna J, Hernandez Chavez G, Soberon X, Bolivar F, Gosset G (2004) Metabolic engineering and protein directed evolution increase the yield of l-phenylalanine synthesized from glucose in Escherichia coli. Biotechnol Bioeng 87:516–524

    Article  PubMed  CAS  Google Scholar 

  2. Becker J, Wittmann C (2012) Bio-based production of chemicals, materials and fuels–Corynebacterium glutamicum as versatile cell factory. Curr Opin Microbiol 23:631–640

    CAS  Google Scholar 

  3. Date M, Itaya H, Matsui H, Kikuchi Y (2006) Secretion of human epidermal growth factor by Corynebacterium glutamicum. Lett Appl Microbiol 42:66–70

    Article  PubMed  CAS  Google Scholar 

  4. Date M, Yokoyama K, Umezawa Y, Matsui H, Kikuchi Y (2003) Production of native-type Streptoverticillium mobaraense transglutaminase in Corynebacterium glutamicum. Appl Environ Microbiol 69:3011–3014

    Article  PubMed  CAS  Google Scholar 

  5. Dueber JE, Wu GC, Malmirchegini GR, Moon TS, Petzold CJ, Ullal AV, Prather KL, Keasling JD (2009) Synthetic protein scaffolds provide modular control over metabolic flux. Nat Biotechnol 27:753–759

    Article  PubMed  CAS  Google Scholar 

  6. Eggeling L, Bott M (2005) Handbook of Corynebacterium glutamicum. CRC Press, Baton Rouge

  7. Gelfand DH, Steinberg RA (1977) Escherichia coli mutants deficient in the aspartate and aromatic amino acid aminotransferases. J Bacteriol 130:429–440

    PubMed  CAS  Google Scholar 

  8. Gerigk MR, Maass D, Kreutzer A, Sprenger G, Bongaerts J, Wubbolts M, Takors R (2002) Enhanced pilot-scale fed-batch l-phenylalanine production with recombinant Escherichia coli by fully integrated reactive extraction. Bioprocess Biosyst Eng 25:43–52

    Article  PubMed  CAS  Google Scholar 

  9. Gopinath V, Murali A, Dhar KS, Nampoothiri KM (2012) Corynebacterium glutamicum as a potent biocatalyst for the bioconversion of pentose sugars to value-added products. Appl Microbiol Biotechnol 93:95–106

    Article  PubMed  Google Scholar 

  10. Gu P, Yang F, Kang J, Wang Q, Qi Q (2012) One-step of tryptophan attenuator inactivation and promoter swapping to improve the production of l-tryptophan in Escherichia coli. Microb Cell Fact 11:30–38

    Article  PubMed  CAS  Google Scholar 

  11. Henderson JW, Ricker RD, Bidlingmeyer BA et al (2000) Rapid, accurate, sensitive, and reproducible HPLC analysis of amino acids. USA (Agilent App Note 5980–1193E). Agilent Technologies, Santa Clara

  12. Hsu SK, Lin LL, Lo HH, Hsu WH (2004) Mutational analysis of feedback inhibition and catalytic sites of prephenate dehydratase from Corynebacterium glutamicum. Arch Microbiol 181:237–244

    Article  PubMed  CAS  Google Scholar 

  13. Hu C, Jiang P, Xu J, Wu J, Huang W (2003) Mutation analysis of the feedback inhibition site of phenylalanine-sensitive 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase of Escherichia coli. J Basic Microbiol 43:399–406

    Article  PubMed  CAS  Google Scholar 

  14. Hudson GS, Davidson BE (1984) Nucleotide sequence and transcription of the phenylalanine and tyrosine operons of Escherichia coli K12. J Mol Biol 180:1023–1051

    Article  PubMed  CAS  Google Scholar 

  15. Ikeda M (2006) Towards bacterial strains overproducing l-tryptophan and other aromatics by metabolic engineering. Appl Microbiol Biotechnol 69:615–626

    Article  PubMed  CAS  Google Scholar 

  16. Ikeda M, Katsumata R (1992) Metabolic engineering to produce tyrosine or phenylalanine in a tryptophan-producing Corynebacterium glutamicum Strain. Appl Environ Microbiol 58:781–785

    PubMed  CAS  Google Scholar 

  17. Ikeda M, Ozaki A, Katsumata R (1993) Phenylalanine production by metabolically engineered Corynebacterium glutamicum with the pheA gene of Escherichia coli. Appl Microbiol Biotechnol 39:318–323

    Article  PubMed  CAS  Google Scholar 

  18. Jakoby M, Ngouoto-Nkili CE, Burkovski A (1999) Construction and application of new Corynebacterium glutamicum vectors. Biotechnol Technol 13:437–441

    Article  CAS  Google Scholar 

  19. Juminaga D, Baidoo EEK, Redding-Johanson AM, Batth TS, Burd H, Mukhopadhyay A, Petzold CJ, Keasling JD (2011) Modular Engineering of l-Tyrosine Production in Escherichia coli. Appl Environ Microbiol 78:89–98

    Article  PubMed  Google Scholar 

  20. Kikuchi Y, Date M, Yokoyama K, Umezawa Y, Matsui H (2003) Secretion of active-form Streptoverticillium mobaraense transglutaminase by Corynebacterium glutamicum: processing of the pro-transglutaminase by a cosecreted subtilisin-like protease from Streptomyces albogriseolus. Appl Environ Microbiol 69:358–366

    Article  PubMed  CAS  Google Scholar 

  21. Li PP, Liu YJ, Liu SHJ (2009) Genetic and biochemical identification of the chorismate mutase from Corynebacterium glutamicum. Microbiology 155:3382–3391

    Article  PubMed  CAS  Google Scholar 

  22. Liu DX, Fan CS, Tao JH, Liang GX, Gao SE, Wang HJ, Li X, Song DX (2004) Integration of E. coli aroG-pheA tandem genes into Corynebacterium glutamicum tyrA locus and its effect on l-phenylalanine biosynthesis. World J Gastroenterol 10:3683–3687

    PubMed  CAS  Google Scholar 

  23. Nešvera J, Pátek M (2011) Tools for genetic manipulations in Corynebacterium glutamicum and their applications. Appl Microbiol Biotechnol 90:1641–1654

    Article  PubMed  Google Scholar 

  24. Shu CH, Liao CC (2002) Optimization of l-phenylalanine production of Corynebacterium glutamicum under product feedback inhibition by elevated oxygen transfer rate. Biotechnol Bioeng 77:131–141

    Article  PubMed  CAS  Google Scholar 

  25. Sprenger G (2007) Aromatic amino acids. In: Wendisch VF (ed) Amino acid biosynthesis pathways regulation and metabolic engineering. Springer, Berlin, pp 93–127

    Chapter  Google Scholar 

  26. Takors R, Bathe B, Rieping M, Hans S, Kelle R, Huthmacher K (2007) Systems biology for industrial strains and fermentation processes-example: amino acids. J Biotechnol 129:181–190

    Article  PubMed  CAS  Google Scholar 

  27. Tribe D, Camakaris H, Pittard J (1976) Constitutive and repressivle enzymes of the common pathway of aromatic biosynthesis in Escherichia coli K-12: regulation of enzyme synthesis at different growth rates. J Bacteriol 127:1085

    PubMed  CAS  Google Scholar 

  28. Tyo KE, Kocharin K, Nielsen J (2010) Toward design-based engineering of industrial microbes. Curr Opin Microbiol 13:255–262

    Article  PubMed  CAS  Google Scholar 

  29. Wu YQ, Jiang PH, Fan CS, Wang JG, Shang L, Huang WD (2003) Co-expression of five genes in E. coli or l-phenylalanine in Brevibacterium flavum. World J Gastroentero 9:342–346

    CAS  Google Scholar 

  30. Xu DQ, Tan YZ, Huan XJ, Hu XQ, Wang XY (2010) Construction of a novel shuttle vector for use in Brevibacterium flavum, an industrial amino acid producer. J Microbiol Method 80:86–92

    Article  CAS  Google Scholar 

  31. Yakandawala N, Romeo T, Friesen AD, Madhyastha S (2008) Metabolic engineering of Escherichia coli to enhance phenylalanine production. Appl Microbiol Biotechnol 78:283–291

    Article  PubMed  CAS  Google Scholar 

  32. Yin L, Hu X, Xu D, Ning J, Chen J, Wang X (2012) Co-expression of feedback-resistant threonine dehydratase and acetohydroxy acid synthase increase l-isoleucine production in Corynebacterium glutamicum. Metab Eng 14:542–550

    Article  PubMed  CAS  Google Scholar 

  33. Zhou H, Liao X, Wang T, Du G, Chen J (2010) Enhanced l-phenylalanine biosynthesis by co-expression of pheA fbr and aroF wt. Bioresour Technol 101:4151–4156

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

We thank Professor Byong Lee at Jiangnan University for his discussion and revision. This work was financially supported by the Key Program of National Natural Science Foundation of China (31130043), the National Natural Science Foundation of China (31200020, 31000054, 31171638), the Priority Academic Program Development of Jiangsu Higher Education Institutions, the Jiangsu Planned Projects for Postdoctoral Research Funds (1101053C) and the Independent Innovation Program of Jiangnan University (JUSRP111A23).

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Authors and Affiliations

  1. The Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi, 214122, China

    Chuanzhi Zhang, Junli Zhang, Zhen Kang, Xiaobin Yu & Tianwen Wang

  2. School of Biotechnology, Jiangnan University, Wuxi, 214122, China

    Chuanzhi Zhang, Junli Zhang, Zhen Kang, Guocheng Du, Xiaobin Yu, Tianwen Wang & Jian Chen

  3. National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi, 214122, China

    Jian Chen

  4. The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi, 214122, China

    Guocheng Du

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  1. Chuanzhi Zhang
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  2. Junli Zhang
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  3. Zhen Kang
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Correspondence to Zhen Kang or Jian Chen.

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Zhang, C., Zhang, J., Kang, Z. et al. Enhanced production of l-phenylalanine in Corynebacterium glutamicum due to the introduction of Escherichia coli wild-type gene aroH . J Ind Microbiol Biotechnol 40, 643–651 (2013). https://doi.org/10.1007/s10295-013-1262-x

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  • DOI: https://doi.org/10.1007/s10295-013-1262-x

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