Developments in genetic engineering of novel acetogens

The development of processes for sustainable energy and chemical production is of great importance for the health of our planet. Utilising suitable feedstocks such as renewable resources and existing waste streams is central to making such processes a reality. Microorganisms can be employed in the processing of a diverse range of such feedstocks, offering unique routes of production for useful and valuable chemical products. Acetogenic organisms, capable of fermenting single carbon (C1) feedstocks are especially interesting from the perspective of industrial application. Their natural metabolism and biochemistry enables fixation of low energy C1 compounds, under conditions which would typically be unfeasible with traditional chemical catalysts. Genetic research and development of new acetogenic species opens the door to industrially feasible, and economically attractive, sustainable bioprocessing. This study outlines the genetic development of the methanol and syngas fermenting acetogen E. limosum. This includes establishing gene-transfer and genetic engineering tools in this organism for first time. Additionally, we demonstrate the genetic engineering of synthetic metabolic pathways in this strain to enable production of valuable chemicals, acetone and isopropanol. Gene-transfer is a cornerstone of modern genetic research in microorganisms, and so effective methods of establishing it are of significant value. We present the development of an improved methodology for enabling and enhancing gene transfer in recalcitrant microorganisms which contain active restriction modification (RM) systems. The method harnesses lambda-red recombineering to support the rapid creation of tailored methylation donor (MD) strains for the preparation and protection of transforming plasmids. The process is uniquely designed in a manner which enables compatibility with both electroporation and conjugation methods of gene transfer