Biotechnical Production of Ethylene in S. Cerevisiae - Insights from Metabolic Modeling, Cultivation Studies and Enzyme Engineering

In the last decades the environmental, production and social sustainability of the oil based industry has become increasingly questioned. Major efforts are hence being put into place to find alternative production methods for traditional petroleum derived products. For the fuel sector several bio-alternatives are being developed, however approximately 15 % of all oil is used to produce petrochemicals other than fuels and bio-production methods of these are rare. One of the major petrochemicals is ethylene (ethene), a hydrocarbon which due to its chemical structure makes it susceptible to a wide range of chemical conversions. In nature ethylene is most known as a plant hormone. However, there are also microorganisms which produce ethylene, foremost plant pathogens and soil bacteria. There are hence biological pathways which can be utilized for the production of ethylene. Through metabolic modelling it was concluded that the bacterial pathway employing the ethylene forming enzyme (EFE) is the most promising when S. cerevisiae is employed as the host organism. The overall aim of this project was to elucidate the functionality of this cell factory. In our previous study we showed that a good or increased provision of the EFE substrates oxygen and 2-oxoglutarate is positive for ethylene productivity. However, increased provision of the EFE-substrate arginine contrary to our expectations reduced ethylene formation. To study the effect of arginine on the system further, we employ two metabolic strategies to reduce intracellular arginine availability; overexpression of CAR1 and deletion of ARG4. Through metabolic network reconstruction of the EFE reaction together with the S. cerevisiae metabolism and subsequent flux balance analysis of the system, we have further identified several other targets predicted to increase ethylene formation. Three candidates from the list – ALT1, CIT1 and IDP2 – were overexpressed and their effect on ethylene productivity in vivo evaluated. Our current research is aimed at developing a deeper understanding of the enzyme functionality. Structure determination of the EFE has proven difficult, however through sequence alignment of three EFE, of which one had lost the ethylene formation capability, 10 amino acid residues emerged which are seemingly important for the capability of the enzyme to form ethylene. Taken together this work elucidates the possibilities and challenges of turning S. cerevisiae into a cell factory for ethylene production. This research is funded by the EU 7^th Framework Programme under the grant agreement n°FP7-241566 - BIOCORE.