2-Butanol and Butanone Production in Saccharomyces cerevisiae through Combination of a B12 Dependent Dehydratase and a Secondary Alcohol Dehydrogenase Using a TEV-Based Expression System

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.

Tobacco Etch Virus protease: A shortcut across biotechnologies

“…TEVp has been often used in vivo to facilitate stoichiometric expression of multiples genes in bacteria (Chen et al, 2010), yeast (Ghiaci et al, 2014), plants (Majer et al, 2015) and mammalian cells (Cesaratto et al, 2015). Using this strategy it is possible to achieve expression of protein subunits at stoichiometric equal levels and at the same physical location.…”

“…Using this strategy it is possible to achieve expression of protein subunits at stoichiometric equal levels and at the same physical location. For instance, the equimolar expression of three subunits of diol dehydratase complex in the cytosol of yeasts (Ghiaci et al, 2014) and of the two chains of IgG in the ER of mammalian cells (Cesaratto et al, 2015). Different proteins can be packaged in a single polyprotein together with TEVp, mimicking the successful processing of precursor polypeptide during TEV infection (Chen et al, 2010).…”

An Engineered Glycerol Dehydratase With Improved Activity for the Conversion of meso‐2,3‐butanediol to Butanone

“…Microbial biosynthesis of butanone has been demonstrated in Escherichia coli , Saccharomyces cerevisiae , and Klebsiella pneumoniae . In each of these cases, the critical step is the dehydration of 2,3‐butanediol (produced by native or engineered metabolic pathways) to butanone. There are no known enzymes that natively carry out this reaction.…”

Discovery and Evaluation of Biosynthetic Pathways for the Production of Five Methyl Ethyl Ketone Precursors

“…26 MEK shows superior characteristics compared to conventional gasoline and ethanol in terms of its thermo-physical properties, increased combustion stability at low engine load, and cold boundary conditions, while decreasing particle emissions. 27 There is no known native microbial producer of MEK, but in the recent studies this molecule was produced in E. coli 28,29 and S. cerevisiae 6 by introducing novel biosynthetic pathways. To convert 2,3-butanediol to MEK, Yoneda et al 30 introduced into E. coli a B-12 dependent glycerol dehydratase from Klebsiella pneumoniae.…”

“…Srirangan et al 29 expressed in E. coli a set of promiscuous ketothiolases from Cupriavidus necator to form 3-ketovaleryl-CoA, and they further converted this molecule to MEK by expressing acetoacetyl-CoA:acetate/butyrate:CoA transferase and acetoacetate decarboxylase from Clostridium acetobutylicum. In S. cerevisiae, Ghiaci et al 6 expressed a B12-dependent diol dehydratase from Lactobacillus reuteri to convert 2,3-butanediol to MEK. Alternatively, hybrid biochemical/chemical approaches were proposed where precursors of MEK were biologically produced through fermentations and then catalytic processes were used to produce MEK.…”