title: A novel arabinose-inducible genetic operation system developed for Clostridium cellulolyticum.
abstract: BACKGROUND
  Clostridium cellulolyticum and other cellulolytic Clostridium strains are natural producers of lignocellulosic biofuels and chemicals via the consolidated bioprocessing (CBP) route, and systems metabolic engineering is indispensable to meet the cost-efficient demands of industry. Several genetic tools have been developed for Clostridium strains, and an efficient and stringent inducible genetic operation system is still required for the precise regulation of the target gene function.
  
  
  RESULTS
  Here, we provide a stringent arabinose-inducible genetic operation (ARAi) system for C. cellulolyticum, including an effective gene expression platform with an oxygen-independent fluorescent reporter, a sensitive MazF-based counterselection genetic marker, and a precise gene knock-out method based on an inducible ClosTron system. A novel arabinose-inducible promoter derived from Clostridium acetobutylicum is employed in the ARAi system to control the expression of the target gene, and the gene expression can be up-regulated over 800-fold with highly induced stringency. The inducible ClosTron method of the ARAi system decreases the off-target frequency from 100% to 0, which shows the precise gene targeting in C. cellulolyticum. The inducible effect of the ARAi system is specific to a universal carbon source L-arabinose, implying that the system could be used widely for clostridial strains with various natural substrates.
  
  
  CONCLUSIONS
  The inducible genetic operation system ARAi developed in this study, containing both controllable gene expression and disruption tools, has the highest inducing activity and stringency in Clostridium by far. Thus, the ARAi system will greatly support the efficient metabolic engineering of C. cellulolyticum and other mesophilic Clostridium strains for lignocellulose bioconversion.
authors.affiliation: Shandong Provincial Key Laboratory of Energy Genetics, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, 189, Songling Road, Qingdao, 266101 People's Republic of China ; Qingdao Engineering Laboratory of Single Cell Oil, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, 189, Songling Road, Qingdao, 266101 People's Republic of China ; University of Chinese Academy of Sciences, Chinese Academy of Sciences, 19, Yuquan Road, Beijing, 100049 People's Republic of China.
authors.forename: Jie
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Shandong Provincial Key Laboratory of Energy Genetics, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, 189, Songling Road, Qingdao, 266101 People's Republic of China ; Qingdao Engineering Laboratory of Single Cell Oil, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, 189, Songling Road, Qingdao, 266101 People's Republic of China.
authors.forename: Ya-Jun
authors.initials: YJ
authors.lastname: Liu
authors.affiliation: Shandong Provincial Key Laboratory of Energy Genetics, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, 189, Songling Road, Qingdao, 266101 People's Republic of China ; Qingdao Engineering Laboratory of Single Cell Oil, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, 189, Songling Road, Qingdao, 266101 People's Republic of China.
authors.forename: Gu-Zhen
authors.initials: GZ
authors.lastname: Cui
authors.affiliation: Shandong Provincial Key Laboratory of Energy Genetics, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, 189, Songling Road, Qingdao, 266101 People's Republic of China ; Key Laboratory of Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, 189, Songling Road, Qingdao, 266101 People's Republic of China ; Qingdao Engineering Laboratory of Single Cell Oil, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, 189, Songling Road, Qingdao, 266101 People's Republic of China.
authors.forename: Qiu
authors.initials: Q
authors.lastname: Cui
keywords: Consolidated bioprocessing; Counterselection marker; Inducible gene expression; Metabolic engineering; Targetron