Production of 2,3-butanediol in Saccharomyces cerevisiae by in silico aided metabolic engineering.

Department of Chemical & Biological Engineering, Korea University, Seoul, 136-701, Republic of Korea. .
Microbial Cell Factories (Impact Factor: 4.25). 05/2012; 11:68. DOI: 10.1186/1475-2859-11-68
Source: PubMed

ABSTRACT 2,3-Butanediol is a chemical compound of increasing interest due to its wide applications. It can be synthesized via mixed acid fermentation of pathogenic bacteria such as Enterobacter aerogenes and Klebsiella oxytoca. The non-pathogenic Saccharomyces cerevisiae possesses three different 2,3-butanediol biosynthetic pathways, but produces minute amount of 2,3-butanediol. Hence, we attempted to engineer S. cerevisiae strain to enhance 2,3-butanediol production.
We first identified gene deletion strategy by performing in silico genome-scale metabolic analysis. Based on the best in silico strategy, in which disruption of alcohol dehydrogenase (ADH) pathway is required, we then constructed gene deletion mutant strains and performed batch cultivation of the strains. Deletion of three ADH genes, ADH1, ADH3 and ADH5, increased 2,3-butanediol production by 55-fold under microaerobic condition. However, overproduction of glycerol was observed in this triple deletion strain. Additional rational design to reduce glycerol production by GPD2 deletion altered the carbon fluxes back to ethanol and significantly reduced 2,3-butanediol production. Deletion of ALD6 reduced acetate production in strains lacking major ADH isozymes, but it did not favor 2,3-butanediol production. Finally, we introduced 2,3-butanediol biosynthetic pathway from Bacillus subtilis and E. aerogenes to the engineered strain and successfully increased titer and yield. Highest 2,3-butanediol titer (2.29 g·l-1) and yield (0.113 g·g-1) were achieved by Δadh1 Δadh3 Δadh5 strain under anaerobic condition.
With the aid of in silico metabolic engineering, we have successfully designed and constructed S. cerevisiae strains with improved 2,3-butanediol production.

  • Source
    [Show abstract] [Hide abstract]
    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.
    PLoS ONE 07/2014; 9(7):e102774. · 3.53 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Volatile organic compounds (VOCs) produced during microbial fermentations determine the flavor of fermented food and are of interest for the production of fragrances or food additives. However, the microbial synthesis of these compounds from simple carbon sources has not been well investigated so far. Here, we analyzed the headspace over glucose minimal salt medium cultures of Saccharomyces cerevisiae using multi-capillary column-ion mobility spectrometry (MCC-IMS). The high sensitivity and fast data acquisition of the MCC-IMS enabled online analysis of the fermentation off-gas and 19 specific signals were determined. To four of these volatile compounds, we could assign the metabolites ethanol, 2-pentanone, isobutyric acid, and 2,3-hexanedione by MCC-IMS measurements of pure standards and cross validation with thermal desorption–gas chromatography-mass spectrometry measurements. Despite the huge biochemical knowledge of the biochemistry of the model organism S. cerevisiae, only the biosynthetic pathways for ethanol and isobutyric acid are fully understood, demonstrating the considerable lack of research of volatile metabolites. As monitoring of VOCs produced during microbial fermentations can give valuable insight into the metabolic state of the organism, fast and non-invasive MCC-IMS analyses provide valuable data for process control.
    Metabolites. 09/2014; 4(3):751-774.
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The effect of temperature on the production of aromatic compounds using Heisei Miyazaki yeast MF062 was compared between 10 industrial yeasts. All yeasts tested produced characteristic patterns of alcohols and esters in fermentation tests with rice-koji at 20, 28 and 38°C. The concentration and composition in mature moromi with rice-koji at 20, 28 and 38°C were almost the same as those with barley-koji. Therefore, it was suggested that fermentation temperature is an important factor in the production of aromatic compounds. MF062 produced almost the same concentration of β-phenethyl alcohol at both 38 and 28°C. The concentration was higher than that generated by the other 10 yeasts. MF062 produced higher concentrations of i-butyl alcohol than the other yeasts at higher fermentation temperatures. Moreover, compared with the other yeasts, MF062 produced a lower concentration of acetate, which can give an off-flavour in excess concentrations in shochu. The production of acetoin was divided into two groups – a high producing group and a low producing group – at all temperatures. MF062 belonged to the latter group and showed preferred characteristics in the production of shochu, resulting in a high concentration of preferred aromatic compounds and a low concentration of compounds that impart an off-flavour. Copyright © 2013 The Institute of Brewing & Distilling
    Journal of the Institute of Brewing. 12/2012; 118(4).


Available from
May 21, 2014