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, Moorella thermoacetica, partly due to the lack of suitable shuttle vectors. However, M. thermoacetica 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 M. thermoacetica 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.e., ethanol production in this organism. The non-native ethanologenesis in M. thermoacetica was achieved via plasmid-borne overexpression of the native aldh gene and heterologous expression of Clostridium autoethanogenum adhE1 gene. This result demonstrates the importance of the developed replicating plasmid vector for genetic and metabolic engineering efforts in industrially important M. thermoacetica.
The platform chemical
ethylene glycol (EG) is used to manufacture
various commodity chemicals of industrial importance, but largely
remains synthesized from fossil fuels. Although several novel metabolic
pathways have been reported for its bioproduction in model organisms,
none has been reported for gas-fermenting, non-model acetogenic chassis
organisms. Here, we describe a novel, synthetic biochemical pathway
to convert acetate into EG in the industrially important gas-fermenting
acetogen,
Clostridium autoethanogenum
. We not only developed a computational workflow to design and analyze
hundreds of novel biochemical pathways for EG production but also
demonstrated a successful pathway construction in the chosen host.
The EG production was achieved using a two-plasmid system to bypass
unfeasible expression levels and potential toxic enzymatic interactions.
Although only a yield of 0.029 g EG/g fructose was achieved and therefore
requiring further strain engineering efforts to optimize the designed
strain, this work demonstrates an important proof-of-concept approach
to computationally design and experimentally implement fully synthetic
metabolic pathways in a metabolically highly specific, non-model host
organism.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.