Fine-tuning expression of key enzymes is an important aspect of synthetic biology, which determines the capability of artificial gene regulation. Clostridium acetobutylicum is a potential cell factory due to its capability of utilizing multiple sugars and producing a variety of chemicals.

For genetic manipulation of C. acetobutylicum, gene knockout methods based on homologous recombination using replicative/non-replicative vectors or Group II intron retrotransposition have been well established. However, a stringent, effective, and convenient-to-use inducible gene expression system that can be used for regulating the gene expression strength in C. acetobutylicum is currently not available.

A research group led by Prof. LI Yin and Associate Prof. ZHANG Yanping at the Institute of Microbiology, CAS, developed an anhydrotetracycline-inducible gene expression system for solvent-producing bacterium C. acetobutylicum. This system, designated as pGusA2-2tetO1, consists of a functional chloramphenicol acetyltransferase gene promoter containing tet operators (tetO), Pthl promoter (thiolase gene promoter from C. acetobutylicum) controlling TetR repressor expression cassette and the chemical inducer anhydrotetracycline (aTc).

This pGusA2-2tetO1 system allows regulation of gene expression in an inducer concentration-dependent way, with a maximal inducibility of over two orders of magnitude. The stringency of TetR repression supports the introduction of the lethal genes into C. acetobutylicum. In addition, it can be directly used in the widely used culture media containing glucose. This system can therefore be used in turning genes on/off, regulating gene expression strength, and developing advanced genetic tools in C. acetobutylicum.

The work has been published in Metabolic Engineering, 2012, 14(1): 59-67(doi:10.1016/j.ymben.2011.10.004). The co-first authors of this paper are Dr. DONG Hongjun, and PhD student TAO Wenwen.

Full article can be found at: http://www.sciencedirect.com/science/article/pii/S1096717611001054