Background Ethyl acetate is a bulk chemical traditionally produced via energy intensive chemical esterification. Microbial production of this compound offers promise as a more sustainable alternative process. So far, efforts have focused on using sugar-based feedstocks for microbial ester production, but extension to one-carbon substrates, such as CO and CO2/H2, is desirable. Acetogens present a promising microbial platform for the production of ethyl esters from these one-carbon substrates. Results We engineered the acetogen C. autoethanogenum to produce ethyl acetate from CO by heterologous expression of an alcohol acetyltransferase (AAT), which catalyzes the formation of ethyl acetate from acetyl-CoA and ethanol. Two AATs, Eat1 from Kluyveromyces marxianus and Atf1 from Saccharomyces cerevisiae, were expressed in C. autoethanogenum. Strains expressing Atf1 produced up to 0.2 mM ethyl acetate. Ethyl acetate production was barely detectable (< 0.01 mM) for strains expressing Eat1. Supplementation of ethanol was investigated as potential boost for ethyl acetate production but resulted only in a 1.5-fold increase (0.3 mM ethyl acetate). Besides ethyl acetate, C. autoethanogenum expressing Atf1 could produce 4.5 mM of butyl acetate when 20 mM butanol was supplemented to the growth medium. Conclusions This work offers for the first time a proof-of-principle that autotrophic short chain ester production from C1-carbon feedstocks is possible and offers leads on how this approach can be optimized in the future.

Background Ethyl acetate is a bulk chemical which is traditionally produced via energy intensive chemical esterification. Microbial production of this compound offers promise as a more sustainable alternative. So far, efforts have focused on using sugar-based feedstocks for microbial ester production, but extension to one-carbon substrates, such as CO and CO2/H2, is desirable. Acetogens such as Clostridium autoethanogenum present a promising microbial platform for biochemical production from one-carbon substrates. Results We engineered C. autoethanogenum to produce ethyl acetate from CO by heterologous expression of an alcohol acetyltransferase (AAT) that catalyzes the formation of ethyl acetate from acetyl-CoA and ethanol. Two AATs, Eat1 and Atf1 were expressed in C. autoethanogenum, and ethyl acetate was successfully produced by strains expressing Atf1. Production of ethyl acetate reached 0.2 mM when grown on CO. Supplementation of ethanol was investigated as potential boost for ethyl acetate production but resulted only in a 1.5-fold increase (0.3 mM ethyl acetate). Besides ethyl acetate, C. autoethanogenum expressing Atf1 could produce 4.5 mM of butyl acetate, when butanol was supplemented to the growth medium. Conclusions This work offers for the first time a proof-of-principle that autotrophic short chain ester production from C1 carbon feedstocks is possible and offers leads on how this approach can be optimized in the future.