Sequential (multiple) Gene Knockout in Clostridium Acetobutylicum Using Mobile Group II Intron

Clostridium acetobutylicum is one of the promising microorganisms for the efficient production of renewable chemicals and biofuels. However, due to the lack of efficient genetic manipulation tools, strain improvement has been rather slow. Fortunately, mobile group II intron was successfully applied to gene knockout of Clostridium species, such as Clostridium perfringens (Chen group) and Clostridium acetobutylicum (Minton group and Yang group) in 2005 and 2007, respectively, for the first time. Nevertheless, construction of a multiple gene knockout mutant was not easy, due to the limit of resources for genetic engineering, such as selection marker and temperature-sensitive origin of replication. Since the knockout system based on the mobile group II intron was constructed in a replicable plasmid, curing of the plasmid was required prior to the disruption of the next gene. However, the curing process is not easy, since the replication of the knockout vector is rather stable. We developed a multiple gene-knockout system that does not require marker pop-out process by using a mobile group II intron Ll.ltrBand two different antibiotics markers, erythromycin and thiamphenicol resistance genes. By using this strategy, a quintuple knockout mutant has been developed, recently. We will discuss an improved sequential (multiple) gene knockout strategy. [This work was supported by the Technology Development Program to Solve Climate Changes on Systems Metabolic Engineering for Biorefineries from the Ministry of Science, ICT and Future Planning (MSIP) through the National Research Foundation (NRF) of Korea (NRF-2012M1A2A2026556 and NRF-2012M1A2A2026557); by the C1 Gas Refinery Project funded by the MSIP through the NRF of Korea (2015M3D3A1A01064918); and the Advanced Biomass R&D Center of Korea (2011-0028386) through the Global Frontier Research Program of the MSIP.]