Due to an increasing demand of transportation fuels and a lower availability of crude oils, a gradual shift from oil based fuels towards alternative and renewable fuel resources will be required in the near future. In addition, currently existing renewable alternatives, bioethanol and conventional biodiesel, cannot cover the increasing demand for biofuels and there is therefore a need for new biofuels, like advanced biodiesel, with superior fuel properties. Novel yeast-cell factories will provide production platforms for advanced fuels. Deep cellular understanding and the use of an integrated systems approach are driving the development of several new processes. Here, metabolic pathways leading towards the metabolite acetyl- CoA are crucial pathways for the production of many promising renewable biofuels and chemicals1.
In the first part of this project, Saccharomyces cerevisiae is metabolically engineered to increase the internal carbon flow towards acetyl-CoA and the final biodiesel product, fatty acid ethyl esters (FAEEs). Different metabolic pathways were compared: In the first pathway endogenous alcohol and aldehyde dehydrogenase and a heterologous acetyl-CoA synthetase were over-expressed together with a wax ester synthase (WS) from Marinobacter hydrocarbonoclasticus, responsible for FAEE synthesis. A second alternative pathway uses the heterologous phosphoketolase and acetate kinase, gained from Aspergillus nidulans and several steps from the pentose phosphate pathway (PPP) to reach the intermediate metabolite acetyl-CoA. Several strains were constructed and resulted in a 3 fold improvement for the ethanol degradation pathway (first strategy) and a 1.7 fold improvement for the PHK pathway (second strategy) times improvement of FAEE production in yeast2.
A second part of the project involves the combination of the up-regulated genes from the central carbon metabolism (described above) and up-regulation of acetyl-CoA carboxylase, acyl-CoA-binding protein and NADP-dependent glyceraldehyde-3-phosphate dehydrogenase towards the formation of free fatty acids and eventually FAEEs. Additional strategies for stable and reliable chromosomal integration of the pathways are presented.

Reference:

1. Bouke de Jong, Verena Siewers and Jens Nielsen; Systems biology of yeast: enabling technology for development of cell factories for production of advanced biofuels, Curr Opin Biotechnol (2011), doi:10.1016/j.copbio.2011.11.021

2. Bouke Wim de Jong, Shuobo Shi, Verena Siewers and Jens Nielsen; Improved production of fatty acid ethyl esters in Saccharomyces cerevisiae through up-regulation of the ethanol degradation pathway and expression of the heterologous phosphoketolase pathway, Molecular Cell Factories, submitted.