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

Jie Zhang,Qiu Cui,Ya-Jun Liu,Gu-Zhen Cui

doi:10.1186/s13068-015-0214-2

Abstract

BackgroundClostridium 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.ResultsHere, 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.ConclusionsThe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-015-0214-2) contains supplementary material, which is available to authorized users.

Highlights

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
To obtain enhanced induction activity, we selected the promoter of ptk and the araR regulator expression cassette of C. acetobutylicum to construct the arabinose-inducible genetic operation (ARAi) system (Additional file 1)
Controlled expression of an oxygen-independent green fluorescent protein oxygen-independent fluorescent protein from Pseudomonas putida (PpFbFPm) in C. cellulolyticum using the ARAi system the araR-binding sequences in C. acetobutylicum and C. cellulolyticum were distinct [47], whether the ARAi system could be interfered by the indigenous arabinoseinducible system of C. cellulolyticum was not certain

Summary

Introduction

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. Inducible gene expression tools are required in the metabolic engineering for Clostridium strains because the precise regulation of the target gene function is crucial for either the native pathway engineering or heterologous gene. ClosTron is a gene targeting method derived from a mesophilic mobile group II intron Ll.ltrB [21,22,23] It has been extensively used in the gene disruption of Clostridium strains [24,25,26,27,28], but its high off-targeting activity affects precise genetic engineering [29]. According to the ribozymebased DNA integration mechanism of targetron [30], the DNA targeting specificity can be improved by precise management of the expression of intron RNA and intronencoded protein (IEP) [22,31] using a proper inducible gene expression system

Methods

Results

Discussion

Conclusion