| Summary: | Replicating plasmid shuttle vectors are key tools for efficient genetic and metabolic engineering applications, allowing the development of sustainable bioprocesses using non-model organisms with unique metabolic capabilities. To date, very limited genetic manipulation has been achieved in the thermophilic acetogen, <i>Moorella thermoacetica</i>, partly due to the lack of suitable shuttle vectors. However, <i>M. thermoacetica</i> has considerable potential as an industrial chassis organism, which can only be unlocked if reliable and effective genetic tools are in place. This study reports the construction of a replicating shuttle vector for <i>M. thermoacetica</i> through the identification and implementation of a compatible Gram-positive replicon to allow plasmid maintenance within the host. Although characterisation of plasmid behaviour proved difficult, the designed shuttle vector was subsequently applied for ethanologenesis, <i>i.e.</i>, ethanol production in this organism. The non-native ethanologenesis in <i>M. thermoacetica</i> was achieved via plasmid-borne overexpression of the native <i>aldh</i> gene and heterologous expression of <i>Clostridium autoethanogenum adhE1</i> gene. This result demonstrates the importance of the developed replicating plasmid vector for genetic and metabolic engineering efforts in industrially important <i>M. thermoacetica</i>.
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