Coproduction of Acetaldehyde and Hydrogen during Glucose Fermentation by Escherichia coli

Abstract

Escherichia coli K-12 strain MG1655 was engineered to coproduce acetaldehyde and hydrogen during glucose fermentation by the use of exogenous acetyl-coenzyme A (acetyl-CoA) reductase (for the conversion of acetylCoA to acetaldehyde) and the native formate hydrogen lyase. A putative acetaldehyde dehydrogenase/acetylCoA reductase from Salmonella enterica (SeEutE) was cloned, produced at high levels, and purified by nickel affinity chromatography. In vitro assays showed that this enzyme had both acetaldehyde dehydrogenase activity (68.07 ؎ 1.63 mol min ؊1 mg ؊1 ) and the desired acetyl-CoA reductase activity (49.23 ؎ 2.88 mol min ؊1 mg ؊1 ). The eutE gene was engineered into an E. coli mutant lacking native glucose fermentation pathways (⌬adhE, ⌬ackA-pta, ⌬ldhA, and ⌬frdC). The engineered strain (ZH88) produced 4.91 ؎ 0.29 mM acetaldehyde while consuming 11.05 mM glucose but also produced 6.44 ؎ 0.26 mM ethanol. Studies showed that ethanol was produced by an unknown alcohol dehydrogenase(s) that converted the acetaldehyde produced by SeEutE to ethanol. Allyl alcohol was used to select for mutants with reduced alcohol dehydrogenase activity. Three allyl alcohol-resistant mutants were isolated; all produced more acetaldehyde and less ethanol than ZH88. It was also found that modifying the growth medium by adding 1 g of yeast extract/liter and lowering the pH to 6.0 further increased the coproduction of acetaldehyde and hydrogen. Under optimal conditions, strain ZH136 converted glucose to acetaldehyde and hydrogen in a 1:1 ratio with a specific acetaldehyde production rate of 0.68 ؎ 0.20 g h ؊1 g ؊1 dry cell weight and at 86% of the maximum theoretical yield. This specific production rate is the highest reported thus far and is promising for industrial application. The possibility of a more efficient "no-distill" ethanol fermentation procedure based on the coproduction of acetaldehyde and hydrogen is discussed.

Discovery and Characterization of the 3-Hydroxyacyl-ACP Dehydratase Component of the Plant Mitochondrial Fatty Acid Synthase System

“…The Arabidopsis AtLpxD1 (primers H13 and H14) and AtLpxD2 (primers H15 and H16) genes (the 59 CDS that encodes the putative mitochondrial presequences was removed) were cloned into pBE522 vector (Zhu et al, 2011), resulting in AtlpxD1-pBE and AtlpxD2-pBE. In the presence of AtlpxD1-pBE or AtlpxD2-pBE, the lpxD gene of the E. coli BL21* strain was replaced by a Gen R cassette using a PCR-based method (Datsenko and Wanner, 2000) with primers H17 and H18.…”

Toward aldehyde and alkane production by removing aldehyde reductase activity in Escherichia coli

“…Producing these compounds using microbes would be a renewable alternative to current methods. However, microbial hosts such as E. coli are plagued by many highly active endogenous aldehyde reductases (ALR) that convert aldehydes to alcohols (Rodriguez and Atsumi, 2012; Zhu et al, 2011), which greatly complicates strain engineering for aldehyde production. As a result, E. coli production of only acetaldehyde (Zhu et al, 2011) and isobutyraldehyde (Rodriguez and Atsumi, 2012) has been shown.…”

Evidence that a Metabolic Microcompartment Contains and Recycles Private Cofactor Pools

“…The first reaction converts ethanolamine to acetaldehyde and ammonia and is catalyzed by the B 12 -dependent ethanolamine-ammonia lyase EutBC (7). In the second reaction, the EutE enzyme catalyzes oxidation of acetaldehyde to acetyl-CoA, thereby consuming CoA and reducing NAD ϩ to NADH (7,8). At this point, acetyl-CoA could be shunted directly into the tricarboxylic acid (TCA) cycle to serve as a carbon and energy source (see the horizontal reaction sequence in Fig.…”