Metabolic engineering of Clostridium acetobutylicum for gene essentiality studies

In recent years the need to generate chemicals and fuels by sustainable routes has led to a resurgence of interest in exploiting butanol-producing species Clostridium acetobutylicum. It has a longstanding history in the commercial production of solvents in the so-called Acetone-Butanol-Ethanol (ABE) process. However, it has not operated on a commercial scale for several decades. A better understanding of the physiological factors that affect solvent production could have profound effects on the re-emergence of the ABE fermentation process on an industrial scale. One way to identify those processes of importance in butanol production is through forward genetics using transposon mutagenesis and in particular high-throughput approaches such as Transposon Directed Insection-site Sequencing (TraDIS). The latter enables the identification of essential genes under niche-specific conditions through the simultaneous sequencing of large transposon mutant libraries. Its application in C. acetobutylicum is hindered by poor transformation frequencies. To circumvent this barrier the two endogenous restriction modification systems, CA_C1502 and CA_C3535, were inactivated using allele-coupled exchange (ACE) and a suicide plasmid in a ΔpyrE mutant background of C. acetobutylicum. Their inactivation led to an almost 6-fold increase in transformation frequency compared to the parent strain. Though the improvements in transformation efficiency did not allow the suicide delivery, the use of a replicative conditional vector was demonstrated and a preliminary TraDIS library generated from which a list of 537 potentially essential genes was identified. During implementation of TraDIS, a prophage present in the C. acetobutylicum genome was shown to be active and capable of excision. As phages can have applications as genetic tools, the phage was further investigated. This study showed that the phage’s DNA can be integrated into the chromosome, circularised in the cytoplasm as well as form pseudo-temperate plasmid. The phage was induced with mitomycin C and UV light. Electron microscope characterisation revealed that it has an elongated icosahedral capsid with a long non-contractile tail indicating that it is from the family Siphoviridae. Transduction test and neighbour-joining cladogram study suggest that it is a non-transducing phage. The phage integrase was explored as an integrative tool for application in synthetic biology. This is the first published genetic study of a temperate phage in C. acetobutylicum