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Abstract
2-Butanol and its chemical precursor butanone (methyl ethyl ketone – MEK) are chemicals with potential uses as biofuels and biocommodity chemicals. In order to produce 2-butanol, we have demonstrated the utility of using a TEV-protease based expression system to achieve equimolar expression of the individual subunits of the two protein complexes involved in the B12-dependent dehydratase step (from the pdu-operon of Lactobacillus reuterii), which catalyze the conversion of meso-2,3-butanediol to butanone. We have furthermore identified a NADH dependent secondary alcohol dehydrogenase (Sadh from Gordonia sp.) able to catalyze the subsequent conversion of butanone to 2-butanol. A final concentration of 4±0.2 mg/L 2-butanol and 2±0.1 mg/L of butanone was found. A key factor for the production of 2-butanol was the availability of NADH, which was achieved by growing cells lacking the GPD1 and GPD2 isogenes under anaerobic conditions.
Highlights
Researchers have been contemplating options for substituting fossil fuels for more than a decade
The first step is catalyzed by a B12-dependent diol dehydratase while the second step is catalyzed by a secondary alcohol dehydrogenase (Sadh)
For the dehydrogenation-step we evaluated if the ADH2 gene from Gordonia sp. [25], encoding one of three NADH-dependent secondary alcohol dehydrogenases in this organism, could be a suitable enzyme for butanone to 2-butanol conversion
Summary
Researchers have been contemplating options for substituting fossil fuels for more than a decade. Biofuels made by microorganisms have been among the options with ethanol, fatty acid methyl esters (biodiesel) and different butanol isomers (1-butanol, 2-butanol and iso-butanol) being among the more prominent. The three named isomers of butanol have different physical and chemical characteristics but all of them are considered as high value components either as chemicals or fuel. Various biological routes have been suggested for each of the three butanol isomers, such as amino acid pathways redirection for 1-butanol and isobutanol [2,3,4] and exploitation of the 1-butanol synthesis pathway in clostridium species [5,6,7]. 2-butanol has the highest research octane number and motor octane number among the isomers (110 and 93 respectively) while they all have rather similar heating values (1-butanol: 27, 2-butanol: 26.8, iso-butanol: 26.6 MJ/L) [9] A fourth form of butanol (tert-butanol) exists, but it is solid at room temperature and it is not known to be produced biologically in any organism, it cannot be regarded as a biofuel. 2-Butanol, in particular, holds superior fuel characteristics among the butanol isomers. 2-butanol has the highest research octane number and motor octane number among the isomers (110 and 93 respectively) while they all have rather similar heating values (1-butanol: 27, 2-butanol: 26.8, iso-butanol: 26.6 MJ/L) [9]
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