From Eat to trEat: engineering the mitochondrial Eat1 enzyme for enhanced ethyl acetate production in Escherichia coli

Abstract

Background: Genetic engineering of microorganisms has become a common practice to establish microbial cell factories for a wide range of compounds. Ethyl acetate is an industrial solvent that is used in several applications, mainly as a biodegradable organic solvent with low toxicity. While ethyl acetate is produced by several natural yeast species, the main mechanism of production has remained elusive until the discovery of Eat1 in Wickerhamomyces anomalus. Unlike other yeast alcohol acetyl transferases (AATs), Eat1 is located in the yeast mitochondria, suggesting that the coding sequence contains a mitochondrial pre-sequence. For expression in prokaryotic hosts such as E. coli, expression of heterologous proteins with eukaryotic signal sequences may not be optimal. Results: Unprocessed and synthetically truncated eat1 variants of Kluyveromyces marxianus and Wickerhamomyces anomalus have been compared in vitro regarding enzyme activity and stability. While the specific activity remained unaffected, half-life improved for several truncated variants. The same variants showed better performance regarding ethyl acetate production when expressed in E. coli. Conclusion: By analysing and predicting the N-terminal pre-sequences of different Eat1 proteins and systematically trimming them, the stability of the enzymes in vitro could be improved, leading to an overall improvement of in vivo ethyl acetate production in E. coli. Truncated variants of eat1 could therefore benefit future engineering approaches towards efficient ethyl acetate production.

Synthesis of ethyl acetate from glucose by Kluyveromyces marxianus, Cyberlindnera jadinii and Wickerhamomyces anomalus depending on the induction mode

“…Identification of Eat1 as the key enzyme for bulk synthesis of ethyl acetate and the knowledge that Eat1 is localized in the mitochondria boost the development of genetically modified microbes for optimized ethyl acetate production from sugars [11, 15, 31, 32]. However, microbial conversion of sugar‐rich wastes from the food industry into valuable products (i.e.…”

Metabolic and Process Engineering of Clostridium beijerinckii for Butyl Acetate Production in One Step

“…Similarly, the novel ethyl acetateproducing enzyme Eat1 is also found to be responsible for ethyl acetate production in E. coli, S. cerevisiae, and some other yeasts such as Wickerhamomyces anomalus and Kluyveromyces lactis. 3,5,7,47 From this perspective, it is worth attempting to introduce Eat1 into Clostridium to produce butyl acetate.…”

“…Medium-chain volatile esters with flavors and fruity fragrances are usually value-added in many fields, such as agricultural product processing, food, and pharmacy. , They can be derived by the condensation of alcohols and carboxylic acids using alcohol acetyltransferases (AATase) from microorganisms. − In recent years, a lot of important progress in exploring different chassis, expanding ester types, mining novel enzymes, and developing new processing strategies has been made in microbial ester synthesis. ,,, However, there are few reports on the microbial synthesis of n -butyl acetate. n -Butyl acetate can be naturally found in fruits and plants; it has a fruity aroma and is highly soluble in water.…”

Co-production of hydrogen and ethyl acetate in Escherichia coli

“…From overnight cultures 1–2 mL were transferred to 50 mL modified M9 medium in 250-mL Erlenmeyer flasks and grown at 30 °C and 250 rpm. Strains for anaerobic experiments were inoculated as biological duplicates at an initial OD 600 of 0.2 and incubated at 30 °C and 150 rpm [ 9 ].…”

“…Analysis of ethanol and ethyl acetate in the liquid phase was carried out by an Agilent 7890B gas chromatograph (Agilent, USA) equipped with a flame ionization detector (GC-FID) and an Agilent 7693 autosampler [ 9 ]. Samples were injected into a NukolTM column (30 m × 0.53 mm, 1.0 μm coating, Supelco, USA).…”