Multilevel optimisation of anaerobic ethyl acetate production in engineered Escherichia coli

Abstract

Background Ethyl acetate is a widely used industrial solvent that is currently produced by chemical conversions from fossil resources. Several yeast species are able to convert sugars to ethyl acetate under aerobic conditions. However, performing ethyl acetate synthesis anaerobically may result in enhanced production efficiency, making the process economically more viable. Results We engineered an E. coli strain that is able to convert glucose to ethyl acetate as the main fermentation product under anaerobic conditions. The key enzyme of the pathway is an alcohol acetyltransferase (AAT) that catalyses the formation of ethyl acetate from acetyl-CoA and ethanol. To select a suitable AAT, the ethyl acetate-forming capacities of Atf1 from Saccharomyces cerevisiae, Eat1 from Kluyveromyces marxianus and Eat1 from Wickerhamomyces anomalus were compared. Heterologous expression of the AAT-encoding genes under control of the inducible LacI/ T7 and XylS/ Pm promoters allowed optimisation of their expression levels. Conclusion Engineering efforts on protein and fermentation level resulted in an E. coli strain that anaerobically produced ethyl acetate from glucose at an unprecedented level, i.e. 0.48 C-mol/C-mol or 72% of the maximum pathway yield.

A new approach for balancing the microbial synthesis of ethyl acetate and other volatile metabolites during aerobic bioreactor cultivations

“…Balancing of microbial synthesis of ethyl acetate was so far based on ester concentrations measured in both the liquid and gas phase [3, 8, 19, 20−22, 24, 31]. Here, a new balancing method was developed which is solely based on gas‐phase concentrations.…”

“…Especially yeasts are able to produce this ester in high amounts [3, 7, 8] whereby Wickerhamomyces anomalus [8–13], Cyberlindnera jadinii [8, 14−16] and Kluyveromyces marxianus [7, 8, 17−27] have turned out as the most promising species. Recent identification of ethanol acetyltransferase 1 (Eat1) as the key enzyme for biosynthesis of ethyl acetate in yeasts [13] and the latest findings regarding the reaction kinetics [13] and localization of this enzyme in the mitochondria [8, 28] enable rational metabolic engineering for developing improved producer strains [29−31].…”

“…Ethyl acetate microbially synthesized in aerated bioreactors is quickly transferred from the cultivation medium to the gas phase and then discharged from the reactor by the exhaust gas [7, 8, 17−19, 22−24, 31, 32] which is called gas stripping. Other co‐produced volatile organic compounds (VOCs) such as ethanol [19, 22, 33−37] and acetaldehyde [38] are stripped in the same manner.…”

“…Other co‐produced volatile organic compounds (VOCs) such as ethanol [19, 22, 33−37] and acetaldehyde [38] are stripped in the same manner. Stripping enables an efficient process‐integrated recovery of volatile microbial products [37, 39−41] by saving time and energy, reducing the number of required process stages, avoiding VOC accumulation to inhibitory levels, and reducing the risk of microbial VOC degradation [25, 29, 31, 35, 36].…”

“…Stripping of ethyl acetate from aerated cultivation systems can hardly be prevented but calls for adequate handling. Precise balancing of VOC synthesis in aerated bioreactors was hitherto based on regular measurements of the VOC concentrations in the culture medium as well as the exhaust gas [8, 19, 21, 22, 24, 31]. Exclusive quantification of the stripped ester without considering the dissolved ester [44, 45] is insufficient for determining synthesis rates since this strategy ignores the temporal accumulation of already synthesized but not yet stripped ethyl acetate.…”