Co-production of hydrogen and ethyl acetate in Escherichia coli

Abstract

Abstract Background Ethyl acetate (C 4 H 8 O 2 ) and hydrogen (H 2 ) are industrially relevant compounds that preferably are produced via sustainable, non-petrochemical production processes. Both compounds are volatile and can be produced by Escherichia coli before. However, relatively low yields for hydrogen are obtained and a mix of by-products renders the sole production of hydrogen by micro-organisms unfeasible. High yields for ethyl acetate have been achieved, but accumulation of formate remained an undesired but inevitable obstacle. Coupling ethyl acetate production to the conversion of formate into H 2 may offer an interesting solution to both drawbacks. Ethyl acetate production requires equimolar amounts of ethanol and acetyl-CoA, which enables a redox neutral fermentation, without the need for production of by-products, other than hydrogen and CO 2 . Results We engineered Escherichia coli towards improved conversion of formate into H 2 and CO 2 by inactivating the formate hydrogen lyase repressor ( hycA ), both uptake hydrogenases ( hyaAB , hybBC ) and/or overexpressing the hydrogen formate lyase activator ( fhlA ), in an acetate kinase ( ackA ) and lactate dehydrogenase ( ldhA )-deficient background strain. Initially 10 strains, with increasing number of modifications were evaluated in anaerobic serum bottles with respect to growth. Four reference strains ΔldhAΔackA , ΔldhAΔackA p3-fhlA, ΔldhAΔackAΔhycAΔhyaABΔhybBC and ΔldhAΔackAΔhycAΔhyaABΔhybBC p3-fhlA were further equipped with a plasmid carrying the heterologous ethanol acyltransferase (Eat1) from Wickerhamomyces anomalus and analyzed with respect to their ethyl acetate and hydrogen co-production capacity. Anaerobic co-production of hydrogen and ethyl acetate via Eat1 was achieved in 1.5-L pH-controlled bioreactors. The cultivation was performed at 30 °C in modified M9 medium with glucose as the sole carbon source. Anaerobic conditions and gas stripping were established by supplying N 2 gas. Conclusions We showed that the engineered strains co-produced ethyl acetate and hydrogen to yields exceeding 70% of the pathway maximum for ethyl acetate and hydrogen, and propose in situ product removal via gas stripping as efficient technique to isolate the products of interest.