Molecular Cloning, Nucleotide Sequencing, and Expression of Genes Encoding Alcohol Dehydrogenases From the ThermophileThermoanaerobacter brockiiand the MesophileClostridium beijerinckii

Peretz, Moshe; Bogin, Oren; Tel-Or, Shoshana; Cohen, Aliza; Li, Guangshan; Chen, Jiann-Shin; Burstein, Yigal

doi:10.1006/anae.1997.0083

Cited by 54 publications

(51 citation statements)

References 49 publications

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“…Isopropanol production showed that the conversion rate of SADH from C. beijerinckii from acetone to isopropanol is much higher than that of SADH from T. brockii. Indeed, the alcohol dehydrogenase assay using crude extract showed that SADH from C. beijerinckii has a higher activity for isopropanol formation from acetone than that from T. brockii, consistent with the results of Peretz et al (18). Although a difference in expression levels cannot be ruled out, the SADH from C. beijerinckii is a better choice for isopropanol production under our conditions.…”

Section: Discussionsupporting

confidence: 92%

“…We compared isopropanol production levels from the strains with pTA36 or pTA18. The amino acid sequence of SADH from C. beijerinckii has 76% identity and 86% similarity with that from T. brockii (18). However, TA11(pTA39/pTA18) produced lower concentrations of isopropanol and much higher concentrations of acetone than the strain containing adh from C. beijerinckii(pTA39/pTA36).…”

Section: Resultsmentioning

confidence: 97%

“…Furthermore, we compared the activities of the SADHs (encoded by adh) from C. beijerinckii NRRL B593 (7) and Thermoanaerobacter brockii HTD4 (10) in performing the final step of isopropanol production in E. coli. These two SADHs have previously been expressed and characterized in E. coli (18).…”

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Engineered Synthetic Pathway for Isopropanol Production in Escherichia coli

Hanai

Atsumi

Liao

2007

Appl Environ Microbiol

247

147

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A synthetic pathway was engineered in Escherichia coli to produce isopropanol by expressing various combinations of genes from Clostridium acetobutylicum ATCC 824, E. coli K-12 MG1655, Clostridium beijerinckii NRRL B593, and Thermoanaerobacter brockii HTD4. The strain with the combination of C. acetobutylicum thl (acetyl-coenzyme A [CoA] acetyltransferase), E. coli atoAD (acetoacetyl-CoA transferase), C. acetobutylicum adc (acetoacetate decarboxylase), and C. beijerinckii adh (secondary alcohol dehydrogenase) achieved the highest titer. This strain produced 81.6 mM isopropanol in shake flasks with a yield of 43.5% (mol/mol) in the production phase. To our knowledge, this work is the first to produce isopropanol in E. coli, and the titer exceeded that from the native producers.The search for feasible petroleum substitutes (bioalcohols, biodiesel, and bioplastics) from renewable resources has recently become a global priority as the atmospheric carbon dioxide level rises and petroleum resources become increasingly expensive. Isopropanol is one of the secondary alcohols that can be produced by microbes (17). It could be used as a biofuel to partially replace gasoline. Isopropanol is also used instead of methanol to esterify fat and oil to produce biodiesel, which reduces its tendency to crystallize at low temperatures (11). Finally, isopropanol can be dehydrated to yield propylene (9), which is currently derived from petroleum as a monomer for making polypropylene.Several species of Clostridium have been evaluated for isopropanol production, including 52 strains of Clostridium beijerinckii (4). The maximum production of isopropanol from these strains was 30 mM. Limited knowledge about metabolic regulation of the strains and the difficulty of gene manipulation have hindered further improvements in isopropanol production. On the other hand, Escherichia coli is one of most studied and easily manipulated organisms for metabolic engineering (2, 5, 15). The bacterium has already been shown to produce high titers of ethanol (6) and many other biochemicals (1,2,5,12,22).Bermejo et al.(1) produced acetone in E. coli by introducing four genes from Clostridium acetobutylicum ATCC 824 (thl, ctfAB, and adc encoding acetyl-coenzyme A [CoA] acetyltransferase, acetoacetyl-CoA transferase, and acetoacetate decarboxylase, respectively) under the control of the thl promoter from C. acetobutylicum (1). This engineered E. coli strain produced almost the same level of acetone as C. acetobutylicum ATCC 824 does. However, isopropanol production in E.coli has not been reported. Here, we engineered a synthetic pathway for the production of isopropanol in E. coli.The strategy for the biosynthesis of isopropanol in E. coli utilizes the pathway modeled after C. beijerinckii, which produces isopropanol from acetyl-CoA via acetone (Fig. 1). First, an acetylCoA acetyltransferase condenses two molecules of acetyl-CoA to one molecule of acetoacetyl-CoA (23). Next, an acetoacetyl-CoA transferase transfers CoA from acetoacetyl-CoA to acetate or to bu...

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Section: Discussionsupporting

confidence: 92%

Section: Resultsmentioning

confidence: 97%

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Engineered Synthetic Pathway for Isopropanol Production in Escherichia coli

Hanai

Atsumi

Liao

2007

Appl Environ Microbiol

247

147

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“…Horse-liver ADH can be used for the reduction of a broad range of cyclic ketones and 2-or 3-ketoesters [7], while open-chain methyl and ethyl ketones are the preferred substrates for T. brockii ADH [8]. An NADPH-dependent ADH from Rhodococcus erythropolis (READH) was found that reduces a broad variety of ketones with specific activity, giving (S)-alcohols [9,10]. Furthermore, an NADPHdependent ADH was found in Lactobacillus that converted similar ketone structures but formed (R)-alcohols [11][12][13].…”

Section: Introductionmentioning

confidence: 99%

Alcohol Dehydrogenases as Tools for the Preparation of Enantiopure Metabolites of Drugs with Methyl Alkyl Ketone Moiety

Pękala

2009

Sci. Pharm.

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Three dehydrogenases -(R)-alcohol dehydrogenase from L. kefir, (S)-aromatic alcohol dehydrogenase from T. sp. and (S)-alcohol dehydrogenase from T. brockii -were tested for the preparation of enantiopure hydroxyl metabolites of pentoxifylline (PTX), propentofylline (PPT) and denbufylline (DBF). These metabolites have an important pharmacological significance. The experimental conditions were optimized for biocatalytic reactions. LKADH produced the chiral secondary alcohols: (R)-OHPTX, (R)-OHPPT and (R)-OHDBF, in an antiPrelog's rule configuration. In contrast, TBADH and SAADH also generated chiral secondary alcohols, but according to Prelog's rule, giving (S)-OHPTX, (S)-OHPPT and (S)-OHDBF respectively. All the ADHs tested were characterized by a high enantioselectivity (ees of 99-100%), but the yield of bioconversion was only satisfactory for the reactions performed using LKADH, being in the 96-98% range for PPT and PTX respectively.

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“…Using oligonucleotide primers based on the N-terminal amino acid sequences, we have cloned and sequenced the adh gene encoding the novel primary-secondary ADH (Peretz et al, 1997) and the &A and cffB genes for the CoA-transferase (Toth and Chen, 1997 abstract) from C. beiietinckii NRRL B593. Following the sequencing of the cftA and cffB genes, we have now sequenced and identified the genes encoding acetoacetate decarboxylase (a&) and aldehyde dhydrogenase (ald) (Toth and Chen, unpublished).…”

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Enzymology of acetone-butanol-isopropanol formation. Final technical report, June 1, 1985--July 31, 1997

Chen¹

1998

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Molecular Cloning, Nucleotide Sequencing, and Expression of Genes Encoding Alcohol Dehydrogenases From the ThermophileThermoanaerobacter brockiiand the MesophileClostridium beijerinckii

Cited by 54 publications

References 49 publications

Engineered Synthetic Pathway for Isopropanol Production in Escherichia coli

Engineered Synthetic Pathway for Isopropanol Production in Escherichia coli

Alcohol Dehydrogenases as Tools for the Preparation of Enantiopure Metabolites of Drugs with Methyl Alkyl Ketone Moiety

Enzymology of acetone-butanol-isopropanol formation. Final technical report, June 1, 1985--July 31, 1997

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