Abstract
1. Background. 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.
2. Methods. The Eat1 enzyme was identified by comparing the
transcriptome of Wickerhamomyces anomalus
under producing and non-producing conditions. A fusion protein of Eat1 and Gfp was
made to show by light microscopy in which organelle Eat1 was expressed. The
mitochondrial targeting sequence was identified using bioinformatics
techniques. Eat1 was expressed in E.
coli. Ethyl acetate production was optimized by knocking out byproduct
formation, optimizing the expression level, removal of the targeting sequence,
and in-situ product removal.
3. Results and discussion. We have identified this
elusive enzyme. Eat1 is present in all yeasts known to produce ethyl acetate
(figure 1, left). 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 present (Figure 1,
middle)1. We have shown that the enzyme is located in the
mitochondria, and we have identified the leader sequence responsible for
mitochondrial targeting2.
Expression of Eat1 in E. coli resulted
in ethyl acetate production (figure 1, right)1. We have optimized
anaerobic ethyl acetate production in E. coli by deleting LdhA
and AckA, responsible for lactate and acetate production, respectively. This
increased ethyl acetate production but also gave rise to the accumulation of
pyruvate, indicating the Eat1 activity was insufficient. Eat1 activity was
improved by optimizing the induction level and by removing the mitochondrial
targeting sequence. This reduced production of pyruvate but enhanced the
production of acetate and ethanol. This appeared to be caused by the esterase
activity of Eat1, hydrolysing ethyl acetate. By stripping the ethyl acetate
from the broth, we decreased the time Eat1 could hydrolyse ethyl acetate,
resulting in lower acetate and ethanol production. The final ethyl acetate
yield obtained was 0.7 mol/mol, or 70% of the maximum pathway yield3.
4. Conclusions. By multilevel engineering – bioprospecting the key enzyme, optimizing its
expressing, increasing its activity by protein engineering, knocking out
byproduct formation and by applying in situ product recovery - we were able to
efficiently produce ethyl acetate in E. coli. On paper, the
production costs of ethyl acetate are lower than those of bioethanol.