We developed a novel inducible genetic operation system for Clostridium

Clostridium cellulolyticum and other cellulolytic clostridium strains can degrade lignocellulose and produce lignocellulosic biofuels and chemicals. Our group is working towards the construction of clostridial genetic manipulation platform with high efficiency and feasibility. On the basis of previously developed genetic tools (Cui GZ, et al, J Microbiol Methods, 2012, 89: 201-8.; Mohr G, Hong W, et al, PLoS One 2013, 8:e69032; Cui GZ, Appl Microbiol Biotechnol, 2014, 98: 313-23.), a novel arabinose-inducible genetic operation system has been developed recently, which will greatly promote the metabolic engineering for Clostridium and the industrial utilization of lignocellulosic feedstock. The results have been published in Biotechnology for Biofuels (Zhang J, et al, 2015, 8:36). This is an important process of our group on the study of Clostridium strains.

C. cellulolyticum and other cellulolytic strains can degrade cellulose effectively and produce cellulosic ethanol in one step. Thus, the industrial utilization of lignocellulose via the metabolic engineering of the clostridial strains is of great importance in solving the energy crisis and environmental problem in world-wide range. By far, although several genetic tools have been developed for Clostridium strains, including gene disruption methods via either homologous recombination or intron retrohoming mechanism and heterologous gene expression methods using replicative or integrative plasmids, inducible gene expression tools with high activity and stringency are still required. In this study, we constructed a highly efficient arabinose-inducible gene operation system ARAi. This system includes 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. The arabinose-inducible promoter in ARAi system is derived from Clostridium acetobutylicum. In the presence of the specific inducer L-arabinose, the gene expression level could be up-regulated over 800-fold, which is the highest inducing activity for Clostridium according to the known knowledge. In addition, 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 arabinose-inducible gene operation system ARAi developed in this study has high inducing efficiency and stringency, and ARAi can be applied in both controllable gene expression and precise gene disruption. Therefore, this study provides a useful genetic tool for the systems biology and metabolic engineering of Clostridium, which will stimulate the application of Clostridium in the bioconversion of lignocellulosic substrates. The first author of the paper published in Biotechnology for Biofuels is Jie Zhang, a Ph.D. student in our group, and Professor Qiu Cui and Associate Professor Ya-Jun Liu are the co-corresponding authors.

Based on the obtained results in the studies of mesophilic and thermophilic Clostridium strains, our group has published a review article in Biotechnology Journal (Liu YJ, et al, 2015. doi:10.1002/biot.201400716), in which the mobile group II intron-derived targetron technology and its application in metabolic engineering have been summarized and illustrated. This review demonstrates the resent process of our group in the field of genetic engineering for clostridial strains. The first author of the review article is Associate Professor Ya-Jun Liu and Professor Qiu Cui is the corresponding author.

The studies on Clostridium strains in our is supported by the National Basic Research Program of China (973 Program), the National Natural Science Foundation of China, and the Qingdao Science and Technology Development Plan.

NCBI PubMed 

  1. Jie Zhang, Ya-Jun Liu*, Gu-Zhen Cui and Qiu Cui* (2015) A novel arabinose-inducible genetic operation system developed for Clostridium cellulolyticum. Biotechnol. Biofuels 8, 36. [PMID:25763107] [Full text (Publisher website)]
  2. Ya-Jun Liu, Jie Zhang, Gu-Zhen Cui and Qiu Cui* (2015) Current progress of targetron technology: Development, improvement and application in metabolic engineering. Biotechnol. J. 10(6), 855-865. [PMID: 25735546] [Full text (Publisher website)]