title: Expanding metabolic pathway for de novo biosynthesis of the chiral pharmaceutical intermediate L-pipecolic acid in Escherichia coli.
abstract: BACKGROUND
  The six-carbon circular non-proteinogenic compound L-pipecolic acid is an important chiral drug intermediate with many applications in the pharmaceutical industry. In the present study, we developed a metabolically engineered strain of Escherichia coli for the overproduction of L-pipecolic acid from glucose.
  
  
  RESULTS
  The metabolic pathway from L-lysine to L-pipecolic acid was constructed initially by introducing lysine cyclodeaminase (LCD). Next, L-lysine metabolic flux from glucose was amplified by the plasmid-based overexpression of dapA, lysC, and lysA under the control of the strong trc promoter to increase the biosynthetic pool of the precursor L-lysine. Additionally, since the catalytic efficiency of the key enzyme LCD is limited by the cofactor NAD+, the intracellular pyridine nucleotide concentration was rebalanced by expressing the pntAB gene encoding the transhydrogenase, which elevated the proportion of LCD with bound NAD+and enhanced L-pipecolic acid production significantly. Further, optimization of Fe2+and surfactant in the fermentation process resulted in 5.33 g/L L-pipecolic acid, with a yield of 0.13 g/g of glucose via fed-batch cultivation.
  
  
  CONCLUSIONS
  We expanded the metabolic pathway for the synthesis of the chiral pharmaceutical intermediate L-pipecolic acid in E. coli. Using the engineered E. coli, a fast and efficient fermentative production of L-pipecolic acid was achieved. This strategy could be applied to the biosynthesis of other commercially and industrially important chiral compounds containing piperidine rings.
authors.affiliation: State Key Laboratory of Materials Oriented Chemical Engineering, Nanjing, 211816, People's Republic of China.|College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing, 211816, People's Republic of China.
authors.forename: Hanxiao
authors.initials: H
authors.lastname: Ying
authors.affiliation: State Key Laboratory of Materials Oriented Chemical Engineering, Nanjing, 211816, People's Republic of China.|College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing, 211816, People's Republic of China.
authors.forename: Sha
authors.initials: S
authors.lastname: Tao
authors.affiliation: State Key Laboratory of Materials Oriented Chemical Engineering, Nanjing, 211816, People's Republic of China.|College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing, 211816, People's Republic of China.
authors.forename: Jing
authors.initials: J
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Materials Oriented Chemical Engineering, Nanjing, 211816, People's Republic of China.|College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing, 211816, People's Republic of China.
authors.forename: Weichao
authors.initials: W
authors.lastname: Ma
authors.affiliation: State Key Laboratory of Materials Oriented Chemical Engineering, Nanjing, 211816, People's Republic of China. kqchen@njtech.edu.cn.|College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing, 211816, People's Republic of China. kqchen@njtech.edu.cn.
authors.forename: Kequan
authors.initials: K
authors.lastname: Chen
authors.affiliation: State Key Laboratory of Materials Oriented Chemical Engineering, Nanjing, 211816, People's Republic of China.|College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing, 211816, People's Republic of China.
authors.forename: Xin
authors.initials: X
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Materials Oriented Chemical Engineering, Nanjing, 211816, People's Republic of China.|College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing, 211816, People's Republic of China.
authors.forename: Pingkai
authors.initials: P
authors.lastname: Ouyang
mesh_terms: D000642:Ammonia-Lyases / Q000235:genetics; D061249:Batch Cell Culture Techniques; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics; D005285:Fermentation; D015870:Gene Expression; D005947:Glucose / Q000378:metabolism; D060847:Metabolic Engineering / Q000379:methods*; D053858:Metabolic Networks and Pathways / Q000235:genetics*; D009243:NAD / Q000378:metabolism; D009250:NADP Transhydrogenases / Q000235:genetics; D010875:Pipecolic Acids / Q000737:chemistry / Q000378:metabolism*; D010957:Plasmids; D011401:Promoter Regions, Genetic
chemical_list: D029968:Escherichia coli Proteins; D010875:Pipecolic Acids; D009243:NAD; D009250:NADP Transhydrogenases; C556051:pntB protein, E coli; D000642:Ammonia-Lyases; C109507:lysine cyclodeaminase; C031345:pipecolic acid; D005947:Glucose
keywords: Chiral intermediate biosynthesis; Cofactor engineering; L-Pipecolic acid; Lysine cyclodeaminase; Metabolic engineering