title: Improvement of catechin production in Escherichia coli through combinatorial metabolic engineering.
abstract: Reconstruction of highly efficient biosynthesis pathways is essential for the production of valuable plant secondary metabolites in recombinant microorganisms. In order to improve the titer of green tea catechins in Escherichia coli, combinatorial strategies were employed using the ePathBrick vectors to express the committed catechin pathway: flavanone 3β-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR), and leucoanthocyanidin reductase (LAR). Three F3H, three DFR, and two LAR genes originating from different plant species were selected and synthesized, to create 18 pathway variants to be screened in E. coli. Constructs containing F3H(syn) originally from Camellia sinensis, DFR(syn) from Anthurium andraeanum, C. sinensis, or Fragaria ananass, and LAR(syn) from Desmodium uncinatum (p148, p158 and p168) demonstrated high conversion efficiency with either eriodictyol or naringenin as substrate. A highly efficient construct was created by assembling additional copies of DFR(syn) and LAR(syn) enabling a titer of 374.6 ± 43.6 mg/L of (+)-catechin. Improving the NADPH availability via the ΔpgiΔppc mutation, BLΔpgiΔppc-p148 produced the highest titer of catechin at 760.9 ± 84.3 mg/L. After utilizing a library of scaffolding proteins, the strain BLΔpgiΔppc-p168-759 reached the highest titer of (+)-catechin of 910.9 ± 61.3 mg/L from 1.0 g/L of eriodictyol in batch culture with M9 minimal media. The impact of oxygen availability on the biosynthesis of catechin was also investigated.
authors.affiliation: Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, NY 12180, USA; The SATCM Key Laboratory for New Resources and Quality Evaluation of Chinese, Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China. Electronic address: zhaoshujuan@126.com.
authors.forename: Shujuan
authors.initials: S
authors.lastname: Zhao
authors.affiliation: Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, NY 12180, USA. Electronic address: jonesj12@rpi.edu.
authors.forename: J Andrew
authors.initials: JA
authors.lastname: Jones
authors.affiliation: Department of Biological Sciences, Rensselaer Polytechnic Institute, Troy, NY 12180, USA. Electronic address: lachad2@rpi.edu.
authors.forename: Daniel M
authors.initials: DM
authors.lastname: Lachance
authors.affiliation: Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, NY 12180, USA. Electronic address: namitab86@gmail.com.
authors.forename: Namita
authors.initials: N
authors.lastname: Bhan
authors.affiliation: Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, NY 12180, USA. Electronic address: khalio2@rpi.edu.
authors.forename: Omar
authors.initials: O
authors.lastname: Khalidi
authors.affiliation: Chromadex Inc., 2830 Wilderness Place, Boulder, CO 80301, USA. Electronic address: syleshv@chromadex.com.
authors.forename: Sylesh
authors.initials: S
authors.lastname: Venkataraman
authors.affiliation: The SATCM Key Laboratory for New Resources and Quality Evaluation of Chinese, Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China; The MOE Key Laboratory for Standardization of Chinese Medicines and Medicines, Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, Shanghai 201210, China. Electronic address: ztwang@shutcm.edu.cn.
authors.forename: Zhengtao
authors.initials: Z
authors.lastname: Wang
authors.affiliation: Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, NY 12180, USA; Department of Biological Sciences, Rensselaer Polytechnic Institute, Troy, NY 12180, USA. Electronic address: koffam@rpi.edu.
authors.forename: Mattheos A G
authors.initials: MAG
authors.lastname: Koffas
mesh_terms: D002392:Catechin / Q000096:biosynthesis / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D060847:Metabolic Engineering; D006899:Mixed Function Oxygenases / Q000096:biosynthesis / Q000235:genetics; D009249:NADP / Q000096:biosynthesis / Q000235:genetics; D010940:Plant Proteins / Q000096:biosynthesis / Q000235:genetics
chemical_list: D010940:Plant Proteins; D009249:NADP; D002392:Catechin; D006899:Mixed Function Oxygenases
keywords: Catechin; Copy number balancing; NADPH availability; Pathway optimization; Protein scaffolds; ePathBrick