A recombinant Escherichia coli was engineered to produce the commercially important amino acid l-phenylalanine (l-Phe) using glycerol as the carbon source. Compared to the conventionally used glucose and sucrose, glycerol is a less expensive carbon source. As phenylalanine dehydrogenase (PheDH) activity is involved in the last step of l-Phe synthesis in E. coli, a phenylalanine dehydrogenase gene (phedh) from the thermotolerant Bacillus lentus was cloned into pRSFDuet-1 (pPheDH) and expressed in E. coli BL21(DE3). The resulting clone had a limited ability to produce l-Phe from glycerol, possibly because of a poor glycerol uptake by the cell, or an inability to excrete l-Phe, or both. Therefore, yddG gene encoding an aromatic amino acid exporter and glpF gene encoding a glycerol transport facilitator were coexpressed with the phedh in a reengineered E. coli. In a glycerol medium, the maximum l-Phe production rates of the clones pPY (phedh and yddG genes) and pPYF (phedh, yddG and glpF genes) were 1.4- and 1.8-fold higher than the maximum production rate of the pPheDH clone. The better producing pPYF clone was further evaluated in a 5 l stirred-tank fermenter (37 °C, an aeration rate of 1 vvm, an agitation speed of 400 rpm). In the fermenter, the maximum concentration of l-Phe (366 mg/l) was achieved in a much shorter period compared to in the shake flasks. In the latter, the highest titer of l-Phe was only 76 % of the maximum value attained in the fermenter.
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This research was supported by a grant in the program “Strategic Scholarships for Frontier Research Network for the Joint Ph.D. Program, Thai Doctoral Degree” from the Office of the Higher Education Commission, Thailand, the Chulalongkorn University Graduate Scholarship to commemorate the 72nd anniversary of His Majesty King Bhumibol Adulyadej, and the A1B1-MICO (TRF) project of the Faculty of Science, Chulalongkorn University. Access to some scientific equipment was provided by the National Center of Excellence for Environmental and Hazardous Waste Management, Chulalongkorn University.
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Thongchuang, M., Pongsawasdi, P., Chisti, Y. et al. Design of a recombinant Escherichia coli for producing l-phenylalanine from glycerol. World J Microbiol Biotechnol 28, 2937–2943 (2012). https://doi.org/10.1007/s11274-012-1104-4
- Phenylalanine dehydrogenase
- Glycerol facilitator
- Aromatic amino acid exporter
- Bacillus lentus
- Escherichia coli BL21(DE3)