Design of a recombinant Escherichia coli for producing L-phenylalanine from glycerol.
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.
Program in Biotechnology, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand.
Mayura
M
Thongchuang
Piamsook
P
Pongsawasdi
Yusuf
Y
Chisti
Kanoktip
K
Packdibamrung
D000594:Amino Acid Oxidoreductases / Q000235:genetics / Q000378:metabolism; D026921:Amino Acid Transport Systems, Neutral / Q000235:genetics / Q000378:metabolism; D020346:Aquaporins / Q000235:genetics / Q000378:metabolism; D001407:Bacillus / Q000201:enzymology; D019149:Bioreactors; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D005990:Glycerol / Q000378:metabolism*; D030781:Organisms, Genetically Modified / Q000235:genetics / Q000378:metabolism; D010649:Phenylalanine / Q000096:biosynthesis*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D026921:Amino Acid Transport Systems, Neutral; D020346:Aquaporins; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; C558730:YddG protein, E coli; C078043:GlpF protein, E coli; D010649:Phenylalanine; D000594:Amino Acid Oxidoreductases; C022342:phenylalanine oxidase; D005990:Glycerol