title: High crude violacein production from glucose by Escherichia coli engineered with interactive control of tryptophan pathway and violacein biosynthetic pathway.
abstract: BACKGROUND
  As bacteria-originated crude violacein, a natural indolocarbazole product, consists of violacein and deoxyviolacein, and can potentially be a new type of natural antibiotics, the reconstruction of an effective metabolic pathway for crude violacein (violacein and deoxyviolacein mixture) synthesis directly from glucose in Escherichia coli was of importance for developing industrial production process.
  
  
  RESULTS
  Strains with a multivariate module for varied tryptophan productivities were firstly generated by combinatorial knockout of trpR/tnaA/pheA genes and overexpression of two key genes trpEfbr /trpD from the upstream tryptophan metabolic pathway. Then, the gene cluster of violacein biosynthetic pathway was introduced downstream of the generated tryptophan pathway. After combination of these two pathways, maximum crude violacein production directly from glucose by E. coli B2/pED+pVio was realized with a titer of 0.6±0.01 g L(-1) in flask culture, which was four fold higher than that of the control without the tryptophan pathway up-regulation. In a 5-L bioreactor batch fermentation with glucose as the carbon source, the recombinant E. coli B2/pED+pVio exhibited a crude violacein titer of 1.75 g L(-1) and a productivity of 36 mg L(-1) h(-1), which was the highest titer and productivity reported so far under the similar culture conditions without tryptophan addition.
  
  
  CONCLUSIONS
  Metabolic pathway analysis using 13C labeling illustrated that the up-regulated tryptophan supply enhanced tryptophan metabolism from glucose, whereas the introduction of violacein pathway drew more carbon flux from glucose to tryptophan, thereby contributing to the effective production of crude violacein in the engineered E. coli cell factory.
authors.affiliation: Department of Chemical Engineering, Tsinghua University, Beijing, 10084, China. fangmingyue88@gmail.com.
authors.forename: Ming-Yue
authors.initials: MY
authors.lastname: Fang
authors.affiliation: Department of Chemical Engineering, Tsinghua University, Beijing, 10084, China. chongzhang@mail.tsinghua.edu.cn.
authors.forename: Chong
authors.initials: C
authors.lastname: Zhang
authors.affiliation: School of Life Sciences, Qingdao Agriculture University, Qingdao, 266109, China. yangsong15@hotmail.com.
authors.forename: Song
authors.initials: S
authors.lastname: Yang
authors.affiliation: School of Life Sciences, Qingdao Agriculture University, Qingdao, 266109, China. cuijinyu212@hotmail.com.
authors.forename: Jin-Yu
authors.initials: JY
authors.lastname: Cui
authors.affiliation: Institute of Microbiology, Chinese Academy of Science, Beijing, 10084, China. jpx@mail.tsinghua.edu.cn.
authors.forename: Pei-Xia
authors.initials: PX
authors.lastname: Jiang
authors.affiliation: Institute of Microbiology, Xinjiang Academy of Agricultural Science, Urumqi, 830000, China. loukai02@mail.tsinghua.edu.cn.
authors.forename: Kai
authors.initials: K
authors.lastname: Lou
authors.affiliation: Department of Bioengineering, Tokyo Institute of Technology, Yokohama, 226-8503, Japan. mwachi@bio.titech.ac.jp.
authors.forename: Masaaki
authors.initials: M
authors.lastname: Wachi
authors.affiliation: Department of Chemical Engineering, Tsinghua University, Beijing, 10084, China. xhxing@mail.tsinghua.edu.cn.
authors.forename: Xin-Hui
authors.initials: XH
authors.lastname: Xing
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D018533:Biomass; D002247:Carbon Isotopes / Q000737:chemistry / Q000378:metabolism; D003352:Corynebacterium / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D055786:Gene Knockout Techniques; D005947:Glucose / Q000378:metabolism*; D007211:Indoles / Q000378:metabolism*; D060847:Metabolic Engineering; D005810:Multigene Family; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D014364:Tryptophan / Q000096:biosynthesis*
chemical_list: D001426:Bacterial Proteins; D002247:Carbon Isotopes; D007211:Indoles; D014364:Tryptophan; D005947:Glucose; C063155:violacein