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Journal of Industrial Microbiology and Biotechnology
Vol. 42 (3), 2015, Pages: 477-486

Metabolic pathway engineering for fatty acid ethyl ester production in using stable chromosomal integration

Bouke Jong, Shuobo Shi, Juan Valle-Rodríguez, Verena Siewers, Jens Nielsen

Chalmers University of Technology, Department of Chemical and Biological Engineering, Gothenburg, Sweden.


Fatty acid ethyl esters are fatty acid derived molecules similar to first generation biodiesel (fatty acid methyl esters; FAMEs) which can be produced in a microbial cell factory. Saccharomyces cerevisiae is a suitable candidate for microbial large scale and long term cultivations, which is the typical industrial production setting for biofuels. It is crucial to conserve the metabolic design of the cell factory during industrial cultivation conditions that require extensive propagation. Genetic modifications therefore have to be introduced in a stable manner. Here, several metabolic engineering strategies for improved production of fatty acid ethyl esters in S. cerevisiae were combined and the genes were stably expressed from the organisms’ chromosomes. A wax ester synthase (ws2) was expressed in different yeast strains with an engineered acetyl-CoA and fatty acid metabolism. Thus, we compared expression of ws2 with and without overexpression of alcohol dehydrogenase (ADH2), acetaldehyde dehydrogenase (ALD6) and acetyl-CoA synthetase (acs SE L641P ) and further evaluated additional overexpression of a mutant version of acetyl-CoA decarboxylase (ACC1 S1157A,S659A ) and the acyl-CoA binding protein (ACB1). The combined engineering efforts of the implementation of ws2, ADH2, ALD6 and acs SE L641P , ACC1 S1157A,S659A and ACB1 in a S. cerevisiae strain lacking storage lipid formation (are1Δ, are2Δ, dga1Δ and lro1Δ) and β-oxidation (pox1Δ) resulted in a 4.1-fold improvement compared with sole expression of ws2 in S. cerevisiae.

Keywords: Yeast, Industrial strain, Chromosomal integration, Homologous recombination, Fatty acid ethyl ester (FAEE), Metabolic pathway engineering.

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