Simultaneous production of isopropanol, butanol, ethanol and 2,3-butanediol by Clostridium acetobutylicum ATCC 824 engineered strains

Florent Collas,Wouter Kuit,Ana M López-Contreras,Rémy Marchal,Frederic Monot,Benjamin Clément

doi:10.1186/2191-0855-2-45

Abstract

Isopropanol represents a widely-used commercial alcohol which is currently produced from petroleum. In nature, isopropanol is excreted by some strains of Clostridium beijerinckii, simultaneously with butanol and ethanol during the isopropanol butanol ethanol (IBE) fermentation. In order to increase isopropanol production, the gene encoding the secondary-alcohol dehydrogenase enzyme from C. beijerinckii NRRL B593 (adh) which catalyzes the reduction of acetone to isopropanol, was cloned into the acetone, butanol and ethanol (ABE)-producing strain C. acetobutylicum ATCC 824. The transformants showed high capacity for conversion of acetone into isopropanol (> 95%). To increase isopropanol production levels in ATCC 824, polycistronic transcription units containing, in addition to the adh gene, homologous genes of the acetoacetate decarboxylase (adc), and/or the acetoacetyl-CoA:acetate/butyrate:CoA transferase subunits A and B (ctfA and ctfB) were constructed and introduced into the wild-type strain. Combined overexpression of the ctfA and ctfB genes resulted in enhanced solvent production. In non-pH-controlled batch cultures, the total solvents excreted by the transformant overexpressing the adh, ctfA, ctfB and adc genes were 24.4 g/L IBE (including 8.8 g/L isopropanol), while the control strain harbouring an empty plasmid produced only 20.2 g/L ABE (including 7.6 g/L acetone). The overexpression of the adc gene had limited effect on IBE production. Interestingly, all transformants with the adh gene converted acetoin (a minor fermentation product) into 2,3-butanediol, highlighting the wide metabolic versatility of solvent-producing Clostridia.

Highlights

The limited supply and the negative environmental effects of the use of petroleum-derived fuels and chemicals have stimulated efforts for the development of more environmentally-friendly processes
The coding sequence of the adh gene from C. beijerinckii NRRL B593 was cloned downstream of the promoter sequence of the thiolase gene from C. acetobutylicum ATCC 824 to form pFC002 plasmid (Table 1)
The promoter sequence of the thiolase gene was chosen in order to maximize expression of the adh gene, since the thiolase gene of C. acetobutylicum was reported to be constitutively expressed (Hartmanis and Gatenbeck 1984; Tummala et al 1999; Alsaker and Papoutsakis 2005)

Summary

Introduction

The limited supply and the negative environmental effects of the use of petroleum-derived fuels and chemicals have stimulated efforts for the development of more environmentally-friendly processes In this respect, the fermentation of carbohydrates into acetone, butanol and ethanol (ABE) or isopropanol, butanol and ethanol (IBE) is a promising way for the production of green chemicals and fuels. DIPE displays good fuel properties and could substitute methyl tert-butyl ether (MTBE) as isooctane index enhancer in gasoline composition (Huang and Sorensen 1990). Another important potential application of biologicallyproduced isopropanol is as a precursor for green propylene, which is the second most important chemical intermediate in the petrochemical industry after ethylene

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Discussion

Conclusion