Ethyl acetate production by the elusive alcohol acetyltransferase from yeast

Kruis, Aleksander J.; Levisson, Mark; Mars, Astrid E.; Ploeg, Max van der; Daza, Fernando Garcés; Ellena, Valeria; Kengen, Servé W. M.; Oost, John van der; Weusthuis, Ruud A.

doi:10.1016/j.ymben.2017.03.004

Cited by 136 publications

(228 citation statements)

References 43 publications

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“…2C, Supplementary Table S3B) in 18 h. Under pH-controlled fermentation, EcJW201 achieved 4.09-fold (from 2.24 ± 0.28 to 9.17 ± 0.12 mg/L), 3.75-fold (from 0.04 ± 0.00 to 0.15 ± 0.02 mg/gDCW/h), and 19-fold (from 0.01 ± 0.00 to 0.19 ± 0.00 mg/g glucose) improvement in titer, specific productivity, and yield, respectively, as compared to the strain performance in the high cell density culture. It is interesting to observe that ethyl acetate was first produced then consumed after 10 h, which is likely due to the endogenous esterase of E. coli as observed in a recent study [43]. Different from ethyl acetate, we did not observe ethyl lactate degradation during fermentation, especially after glucose was depleted.…”

Section: Resultssupporting

confidence: 43%

Microbial Biosynthesis of Lactate Esters

Lee

Trinh

2018

Preprint

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21Background. Green organic solvents such as lactate esters have broad industrial applications and 22 favorable environmental profiles. Thus, manufacturing and use of these biodegradable solvents 23 from renewable feedstocks help benefit the environment. However, to date, the direct microbial 24 biosynthesis of lactate esters from fermentable sugars has not yet been demonstrated. 25Results. In this study, we present a microbial conversion platform for direct biosynthesis of lactate 26 esters from fermentable sugars. First, we designed a pyruvate-to-lactate ester module, consisting 27 of a lactate dehydrogenase (ldhA) to convert pyruvate to lactate, a propionate CoA-transferase 28 (pct) to convert lactate to lactyl-CoA, and an alcohol acyltransferase (AAT) to condense lactyl-29CoA and alcohol(s) to make lactate ester(s). By generating a library of five pyruvate-to-lactate 30 ester modules with divergent AATs, we screened for the best module(s) capable of producing a 31 wide range of linear, branched, and aromatic lactate esters with an external alcohol supply. By co-32 introducing a pyruvate-to-lactate ester module and an alcohol (i.e., ethanol, isobutanol) module 33 into a modular Escherichia coli (chassis) cell, we demonstrated for the first time the microbial 34 biosynthesis of ethyl and isobutyl lactate esters directly from glucose. In an attempt to enhance 35 ethyl lactate production as a proof-of-study, we re-modularized the pathway into 1) the upstream 36 module to generate the ethanol and lactate precursors and 2) the downstream module to generate 37 lactyl-CoA and condense it with ethanol to produce the target ethyl lactate. By manipulating the 38 metabolic fluxes of the upstream and downstream modules through plasmid copy numbers, 39 promoters, ribosome binding sites, and environmental perturbation, we were able to probe and 40 alleviate the metabolic bottlenecks by improving ethyl lactate production by 4.96-fold. We found 41 3 that AAT is the most rate limiting step in biosynthesis of lactate esters likely due to its low activity 42 and specificity towards the non-natural substrate lactyl-CoA and alcohols. 43 Conclusions. We have successfully established the biosynthesis pathway of lactate esters from 44 fermentable sugars and demonstrated for the first time the direct fermentative production of lactate 45 esters from glucose using an E. coli modular cell. This study defines a cornerstone for the microbial 46 production of lactate esters as green solvents from renewable resources with novel industrial 47 applications. 48 49 Keywords: ester; lactate ester; ethyl lactate; isobutyl lactate; acetate ester; alcohol acyltransferase; 50 green solvent; modular cell; Escherichia coli. 51 4

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Section: Resultssupporting

confidence: 43%

Microbial Biosynthesis of Lactate Esters

Lee

Trinh

2018

Preprint

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“…In addition, Km Adh7 was found to exhibit activity toward the oxidation of hemiacetal to ethyl acetate, but the absence of this activity in the ∆adh7 strain of CBS 6556 did not produce a measurable reduction in ethyl acetate biosynthesis. It was recently postulated that ester biosynthesis in K. marxianus may also occur through homologs to the medium-chain acyltransferases Eeb1 and Eht1 from S. cerevisiae , the isoamyl acetate-hydrolyzing esterase Iah1, the N -acetyltransferase Sli1 and/or the alcohol- O- acetyltransferase Eat1 [44, 48]. In S. cerevisiae , Eeb1 and Eht1 have limited activity toward the acetylation of ethanol and are most active toward medium-chain acyl-CoAs and alcohols [49].…”

Section: Discussionmentioning

confidence: 99%

“…Overexpression of Iah1 in S. cerevisiae resulted in lower ester titers due to ester hydrolysis, suggesting that the K. marxianus homolog may not contribute to ethyl acetate biosynthesis [50]. A recently published study describes Eat1 as a putative alcohol- O -acetyltransferase capable of producing ethyl acetate [48]. The new genome-editing tools created in this work should enable the future study of the role of N -acetyltransferases and other enzymes in ethyl acetate biosynthesis.…”

Section: Discussionmentioning

confidence: 99%

CRISPR–Cas9-enabled genetic disruptions for understanding ethanol and ethyl acetate biosynthesis in Kluyveromyces marxianus

Löbs

Engel

Schwartz

et al. 2017

Biotechnol Biofuels

102

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Background:The thermotolerant yeast Kluyveromyces marxianus shows promise as an industrial host for the biochemical production of fuels and chemicals. Wild-type strains are known to ferment high titers of ethanol and can effectively convert a wide range of C 5 , C 6 , and C 12 sugars into the volatile short-chain ester ethyl acetate. Strain engineering, however, has been limited due to a lack of advanced genome-editing tools and an incomplete understanding of ester and ethanol biosynthesis. Results:Enabled by the design of hybrid RNA polymerase III promoters, this work adapts the CRISPR-Cas9 system from Streptococcus pyogenes for use in K. marxianus. The system was used to rapidly create functional disruptions to alcohol dehydrogenase (ADH) and alcohol-O-acetyltransferase (ATF) genes with putative function in ethyl acetate and ethanol biosynthesis. Screening of the KmATF disrupted strain revealed that Atf activity contributes to ethyl acetate biosynthesis, but the knockout reduced ethyl acetate titers by only ~15%. Overexpression experiments revealed that KmAdh7 can catalyze the oxidation of hemiacetal to ethyl acetate. Finally, analysis of the KmADH2 disrupted strain showed that the knockout almost completely eliminated ethanol production and resulted in the accumulation of acetaldehyde. Conclusions:Newly designed RNA polymerase III promoters for sgRNA expression in K. marxianus enable a CRISPRCas9 genome-editing system for the thermotolerant yeast. This system was used to disrupt genes involved in ethyl acetate biosynthesis, specifically KmADH1-7 and KmATF. KmAdh2 was found to be critical for aerobic and anaerobic ethanol production. Aerobically produced ethanol supplies the biosynthesis of ethyl acetate catalyzed by KmAtf. KmAdh7 was found to exhibit activity toward the oxidation of hemiacetal, a possible alternative route for the synthesis of ethyl acetate.

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“…In this work, a RDWC that can incorporate both phase separation and chemical reaction into a single unit is used for the coproduction of EA and BA. RD can improve the conversion of EtOH to 99 % to eliminate an EtOH recycling section because the continuous removal of water would help to reduce the remixing effect in the reactive section , . The flow diagram for this process is illustrated in Fig.…”

Section: Reactive Distillation Processmentioning

confidence: 99%

“…Ethyl acetate (EA) and n ‐butyl acetate (BA) are very important organic materials and excellent industrial solvents. They are widely used in areas such as acetate fibers, synthetic rubbers, coatings, and lacquers, and many reports on their production have been published . At present, in current domestic and foreign industrial production, the main method to synthesize EA and BA is through esterification.…”

Section: Introductionmentioning

confidence: 99%

Coproduction of Ethyl Acetate and n‐Butyl Acetate by Using a Reactive Dividing‐Wall Column

Xie

Peng

et al. 2018

Chem Eng & Technol

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The performance of the reactive distillation dividing-wall column for coproduction of ethyl acetate and butyl acetate was experimentally studied. n-Butanol and ethanol are raw reaction materials that react with acetic acid in the reaction zone to produce n-butyl acetate and ethyl acetate, respectively. n-Butyl acetate is not only a product, but also acts to remove water generated by the esterification reactions. The effects of various parameters, such as catalyst loading per stage, reflux ratio, liquid split and molar feed ratios, ethyl acetate/n-butyl acetate purity, pressure drop, and total energy consumption, are investigated. Results show that ethanol could be completely converted and the products could be easily separated, which shows great industrial application potential in the coproduction of ethyl acetate and n-butyl acetate.

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Ethyl acetate production by the elusive alcohol acetyltransferase from yeast

Cited by 136 publications

References 43 publications

Microbial Biosynthesis of Lactate Esters

Microbial Biosynthesis of Lactate Esters

CRISPR–Cas9-enabled genetic disruptions for understanding ethanol and ethyl acetate biosynthesis in Kluyveromyces marxianus

Coproduction of Ethyl Acetate and n‐Butyl Acetate by Using a Reactive Dividing‐Wall Column

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