A novel arabinose-inducible genetic operation system developed for Clostridium cellulolyticum

Biotechnology for Biofuels

Jie Zhang¹  Ya-Jun Liu²  Gu-Zhen Cui²  Qiu Cui² 

[1] University of Chinese Academy of Sciences, Chinese Academy of Sciences, 19, Yuquan Road, Beijing 100049, People’s Republic of China

[2] 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

关键词: Targetron;    Metabolic engineering;    Inducible gene expression;    Counterselection marker;    Consolidated bioprocessing;   

Others  :  1137816
DOI  :  10.1186/s13068-015-0214-2

 received in 2014-10-23, accepted in 2015-01-29,  发布年份 2015

【 摘 要 】

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.

【 授权许可】

   
2015 Zhang et al.; licensee BioMed Central.

【 图 表 】

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