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. 1998 Mar;64(3):1079-85.
doi: 10.1128/AEM.64.3.1079-1085.1998.

Expression of Clostridium acetobutylicum ATCC 824 genes in Escherichia coli for acetone production and acetate detoxification

L L Bermejo  1 , N E Welker, E T Papoutsakis
Affiliations

Expression of Clostridium acetobutylicum ATCC 824 genes in Escherichia coli for acetone production and acetate detoxification

L L Bermejo et al. Appl Environ Microbiol. 1998 Mar.

Abstract

A synthetic acetone operon (ace4) composed of four Clostridium acetobutylicum ATCC 824 genes (adc, ctfAB, and thl, coding for the acetoacetate decarboxylase, coenzyme A transferase, and thiolase, respectively) under the control of the thl promoter was constructed and was introduced into Escherichia coli on vector pACT. Acetone production demonstrated that ace4 is expressed in E. coli and resulted in the reduction of acetic acid levels in the fermentation broth. Since different E. coli strains vary significantly in their growth characteristics and acetate metabolism, ace4 was expressed in three E. coli strains: ER2275, ATCC 11303, and MC1060. Shake flask cultures of MC1060(pACT) produced ca. 2 mM acetone, while both strains ER2275(pACT) and ATCC 11303(pACT) produced ca. 40 mM acetone. Glucose-fed cultures of strain ATCC 11303(pACT) resulted in a 150% increase in acetone titers compared to those of batch shake flask cultures. External addition of sodium acetate to glucose-fed cultures of ATCC 11303(pACT) resulted in further increased acetone titers. In bioreactor studies, acidic conditions (pH 5.5 versus 6.5) improved acetone production. Despite the substantial acetone evaporation due to aeration and agitation in the bioreactor, 125 to 154 mM acetone accumulated in ATCC 11303(pACT) fermentations. These acetone titers are equal to or higher than those produced by wild-type C. acetobutylicum. This is the first study to demonstrate the ability to use clostridial genes in nonclostridial hosts for solvent production. In addition, acetone-producing E. coli strains may be useful hosts for recombinant protein production in that detrimental acetate accumulation can be avoided.

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Figures

FIG. 1
FIG. 1
Cloning steps for the construction of plasmids ppACT and pACT. For each plasmid, the location and direction of transcription for relevant genes are indicated (arrows). Relevant restriction sites are shown. Abbreviations: Amp, ampicillin resistance gene; MLS, erythromycin resistance gene; ori colE1, gram-negative origin of replication; ori pIM13, gram-positive origin of replication; P, thiolase promoter; lacZ, β-galactosidase gene; lacI, lac repressor gene; adc, AADC gene; thl, thiolase gene; and ctf A,B, units A and B, respectively, of the CoAT gene.
FIG. 2
FIG. 2
Evaluation of E. coli ER2275(pACT) (◊ and ⧫), MC1060(pACT) (□ and ▪), and ATCC 11303(pACT) (○ and •) in batch shake flask experiments. Time profiles of cell growth (a), acetone concentration (b), and acetic acid concentration (c) are shown.
FIG. 3
FIG. 3
Effect of culture pH on acetone production and acetate utilization in a bioreactor E. coli ATCC 11303(pACT) aerobic fermentation.
See this image and copyright information in PMC

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