Engineering Cupriavidus Sp. for Ethylene Production Using CRISPR/Cas Systems

The chemoautotrophic bacterium Cupriavidus necator H16 (formerly known as Ralstonia eutropha H16) can utilise H₂ and CO₂ as a sole energy and carbon source to grow under aerobic conditions. It also accumulates large amounts of polyhydroxybutyrate, a polyalkanoate, used to manufacture biodegradable plastics. This microorganism is of great industrial interest, as metabolic engineering could lead to the production of other highly valuable compounds directly from industrial gas waste. One of these compounds is ethylene, global demand for ethylene is expanding at a rapid rate with production predicted to reach 200 million tonnes by 2020. Ethylene is currently produced from stream cracking or dehydrogenation of ethane, both of these produce large amounts of CO_2, which has a deleterious impact on the environment.We must find environmentally friendly alternatives to satisfy our energy requirements. We aim to engineer Cupriavidus sp. as a platform for the production of hydrocarbon-based products such as ethylene. High throughput metabolic engineering requires fast, flexible and user-friendly genome editing, these tools are not currently readily available in Cupriavidus sp. We demonstrate that ethylene can be produced in Cupriavidus metallidurans utilising the efe gene (ethylene forming enzyme) from P. syringae pv. paseolicola, which is sufficient for ethylene production in heterologous hosts. To improve productivity we are developing HTP genomic engineering tools utilising CRISPR/Cas9 and homologous recombineering.