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

PLoS One. 2014 Jul 23;9(7):e102774. doi: 10.1371/journal.pone.0102774. eCollection 2014.

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 reuteri), 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.

MeSH terms

  • Alcohol Oxidoreductases / genetics
  • Alcohol Oxidoreductases / metabolism*
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • Base Sequence
  • Biosynthetic Pathways
  • Blotting, Western
  • Butanols / metabolism*
  • Butanones / metabolism*
  • Endopeptidases / genetics
  • Endopeptidases / metabolism
  • Gene Expression
  • Genetic Engineering / methods
  • Gordonia Bacterium / enzymology
  • Gordonia Bacterium / genetics
  • Industrial Microbiology / methods
  • Limosilactobacillus reuteri / enzymology
  • Limosilactobacillus reuteri / genetics
  • Molecular Sequence Data
  • Operon / genetics
  • Propanediol Dehydratase / genetics
  • Propanediol Dehydratase / metabolism*
  • Reproducibility of Results
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / metabolism*
  • Vitamin B 12 / metabolism

Substances

  • Bacterial Proteins
  • Butanols
  • Butanones
  • 2-butanol
  • Alcohol Oxidoreductases
  • alcohol dehydrogenase (NAD(P)+)
  • Endopeptidases
  • TEV protease
  • Propanediol Dehydratase
  • Vitamin B 12

Grants and funding

The project was financed by Swedish Energy Agency (project no. 32918-1) and Chalmers Energy Area of Advance, for which we are grateful to them. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.