A study of ethylene production via the 2-oxoglutarate dependent pathway in S. cerevisiae

Doctoral thesis, 2014

The detrimental effect of the petroleum industry on the environment combined with the threat of peak oil has driven the exploration for alternative strategies to produce traditional petrochemicals. Biotechnological production could be an alternative, using microorganisms to convert renewable feedstocks into desired products. A microbial based system for production of the traditional petrochemical ethylene has previously been developed through the expression of a bacterial version of the ethylene forming enzyme (EFE), which catalyzes the 2-oxoglutarate dependent ethylene pathway, in the yeast Saccharomyces cerevisiae. This work aims at deepening the understanding of how the EFE functions and investigate the functionality of the S. cerevisiae-EFE cell factory for ethylene production. To this end metabolic modeling, metabolic engineering as well as several cultivation studies have been performed. Alongside this the enzyme has been characterized through structural prediction and enzyme engineering, which has reviled both a structural entity necessary for ethylene forming functionality as well as a number of specific amino acid residues coupled to ethylene formation. Cultivation studies combined with metabolic engineering strategies have shown that balancing of arginine availability is important for optimal ethylene productivity. Further studies have also revealed that maintaining a high oxygenation level is a crucial cultivation factor for optimal ethylene productivity. This can be linked both to the reaction mechanism of the EFE, for which oxygen is a substrate, but also to an increased requirement of NADH re-oxidation when EFE is expressed. It was found that co-expression of heterologous oxidases could help relieve the redox stress and expression of the Aox1 of Histoplasma capsulatum was concluded to increase the ethylene yield with 28 %. To find further metabolic targets for increased ethylene productivity metabolic modeling was performed. The majority of the targets found were involved in supply of the EFE substrate 2-oxogltuarate, however none of the targets evaluated in vivo so far has given any increase in ethylene yields. Through this work important factors for optimal ethylene formation have been revealed, however it has also shown that more work is required before this system is a competitive alternative for ethylene production.