Multilevel engineering of microbial ethyl acetate production.
Anna C. Bohnenkamp1*, Aleksander J. Kruis1, René H. Wijffels1, Astrid E. Mars2, Servé W. M. Kengen1, Ruud A. Weusthuis1
1 Wageningen University, Wageningen, The Netherlands, 2 Wageningen Research, Wageningen, The Netherlands
*Corresponding author: anna.bohnenkamp@wur.nl
This abstract will show our efforts to develop a process for the production of ethyl acetate. Ethyl acetate is produced from fossil resources at 3.5 million tonnes at a total value of $3.7 billion in 2014. To reduce CO2 emissions a biobased process is desired. Yeasts are able to produce high amounts of ethyl acetate from sugars and ethanol. The development of an efficient fermentation process was hampered because the key enzyme was unknown.
We have identified the Eat1 gene in Wickerhamomyces anomalus by comparing the transcriptome of under producing and non-producing conditions. Eat1 is present in all yeasts known to produce ethyl acetate. It has three activities: alcohol acetyl transferase converting ethanol and acetyl-CoA into ethyl acetate, esterase and thioesterase activity. The latter two activities have a negative effect on ethyl acetate production but were strongly suppressed when ethanol was present1. The cellular location of the enzyme was discovered by making a fusion protein with Gfp. We have shown that the enzyme is located in the mitochondria, and we have identified the leader sequence responsible for mitochondrial targeting2.
We have established and optimized anaerobic ethyl acetate production in E. coli by reducing by-product formation and optimizing the expression of Eat1 on gene and protein level. Closely controlling the process conditions in 1.5L bioreactors and applying gas stripping to reduce hydrolysis of ethyl acetate, further improved the process. The final ethyl acetate yield obtained was 0.7 mol/mol, or 70% of the maximum pathway yield3.
By multilevel engineering – bioprospecting the key enzyme, optimizing its expressing, increasing its activity by protein engineering, knocking out byproduct formation and by first rounds of process optimization - we were able to efficiently produce ethyl acetate in E. coli.
References
[1] Kruis, Alex; Levisson, Mark; Mars, Astrid E.; Ploeg, Max van der; Garcés Daza, Fernando; Ellena, Valeria; Kengen, Servé W.M.; Oost, John van der; Weusthuis, Ruud A. (2017) Ethyl acetate production by the elusive alcohol acetyltransferase from yeast. Metabolic Engineering 41. - p. 92 - 101.
[2] Kruis, A. J., Mars, A. E., Kengen, S. W., Borst, J. W., van der Oost, J., & Weusthuis, R. A. (2018). The alcohol acetyltransferase Eat1 is located in yeast mitochondria. Applied and Environmental microbiology, AEM-01640.
[3] Aleksander J. Kruis, Anna C. Bohnenkamp, Bram Nap, Jochem Nielsen, Pieter Mijnhout, Astrid E. Mars, John van der Oost, Servé W.M. Kengen, René H. Wijffels, Ruud A. Weusthuis. 2019. Enhanced ethyl acetate production in Escherichia coli under anaerobic conditions. In preparation.
