Development of a high-throughput screening method for the selection of high alkane-producing Escherichia coli strains

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Ziaur Rahman, Le Minh Bui, Sun Chang Kim

Korea Advanced Institute of Science and Technology, South Korea

Alkanes are the major constituents of gasoline and jet fuel and the demand for alkanes as biofuels is ever increasing. However, the conventional alkane production process based on expensive raw materials (coal, hydrogen and cobalt) increases the overall cost of alkanes. To meet the growing demand for alkanes, efforts have been made to engineer microbial systems for the economical production of alkanes. Nevertheless, the microbial productions of alkanes are far below our satisfaction. To screen and select the high alkane-producing strains with rational engineering and/or directed evolution, a high-throughput strain selection method is needed. Here, we construct an artificial circuit for the selection of high alkane-producing strains. An alkane sensor plasmid (pALK) was constructed, which consists of an alkane responsive promoter, PalkB, a transcriptional regulator of Alcanivorax borkumensis, AlkS, and a green fluorescence protein, GFP as a reporter. In the presence of alkanes, the circuit is turned on by the release of AlkS from the regulatory region of the PalkB promoter. To observe the response of the PalkB promoter to alkanes, Escherichia coli harboring pALK were supplemented with various kinds of alkanes (e.g., C13, C15, and C17) with concentrations ranging from 10 to 100 ppm. The fluorescent intensities increased accordingly with increasing alkane concentration, which means that the alkane sensitive promoter responded in a concentration-dependent manner. For the detection of alkanes produced by engineered strains, the pALK was co-transformed with alkane-producing plasmid (pET-acr-adc) in E. coli. With our artificial circuit, high alkane-producing strains could be screened successfully. Our results suggest that pALK could be used as a reliable tool for the rapid screening of high alkane-producing hosts and to facilitate the process of rapid strain development.