sorbitol required for cell growth and ethanol production
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Sorbitol required for cel l growth and
ethanol production by Zymomonas
mobil is under heat, ethanol and osmotic
stresses
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Content
Introduction
Methods and results
Discussion Conclusions
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Introduction
Zymomonas mobilis is
a facultative anaerobic
Gram-negative
bacterium which can
ferment certain sugars
through a metabolic
pathway producingbioethanol.
Pic.1 Zymomonas mobilis
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Higher sugar uptake rate and ethanol
yield
Lower biomass production
Higher ethanol tolerance
Non-requirement of controlledaddition of oxygen during
fermentation
It is generally recognized as safe(GRAS)
It is amenability to geneticmanipulations
Zymomonas mobilisshowed some advantages:
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In the present study, they disrupted the gfo gene whichencoded for GFOR in Z. mobilis and the disruptant
strain was designated as Z. mobilis gfo. Autorsshowed for the first time that disruption of the gfo generesulted in the reduction of cell growth and ethanolproduction in Z. mobilis under osmotic stress as well asunder heat and ethanol stresses. They also
demonstrated that the addition of sorbitol not onlypromoted cell growth but also increased thefermentation capability of Z. mobilis under all stressconditions tested. Sorbitol also protected cellularproteins from denaturation under stress conditions.
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Methods
1 DNA isolation and disruption of the Z. mobilis gfo gene
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Testing of effect of stresses on the growth of Z. mobilis parentaland disruptant strains
Testing of effect of stresses on ethanol fermentation by Z. mobilisparental and disruptant strains
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RT-qPCR analysis of gene expression under stresses
Protein extraction and SDS-PAGE
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Complementation experiment
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Pic 2. Schematic illustration of PCR-based construction of the gfo gene
disruption.
The gfo gene in Z. mobilis was disrupted by the fusion PCR-based construction
technique. All PCR reactions were performed by using a PCR amplification kit
(Takara Biomedical, Otsu, Japan) 7
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Figure 2. Effect of heat, ethanol, and osmotic stresses on the growth of Z. mobilis
TISTR548 and Z. mobilis gfo. (A) Z. mobilis cells were grown in YP medium and
incubated at 30, 35, 37, and 39C or (B) in YP medium containing 7, 10, and 13 %
(v/v) ethanol, (C) or in YP medium containing 200, 250 and 300 g/L sucrose.
Effect of stresses on growth of Z. mobilis parental and disruptant
strains
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Table 1.Specific growth rate of Z. mobilis TISTR548 and Z.mobilis gfounder heat,
ethanol and osmotic stresses
The lowest specific growth rates were found in Z. mobilis gfogrowing under the
extreme stress conditions, (at 39C or 13% (v/v) ethanol or 300 gL-sucrose,which
were 1.89-, 2.84-, and 2.09-fold lower than those of Z. mobilis TISTR548 growing
under the same conditions, respectively).
Effect of stresses on growth of Z. mobilis parental and disruptant strains
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Effect of stresses on ethanol production by Z. mobilis parental
and disruptant strains
Table 2. Effect of heat, ethanol and osmotic stresses on ethanol production by
Z. mobilis TISTR548 and Z. mobilis gfo 10
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Real-time RT-PCR analysis of pdc, adhA, and adhB
genes under stress conditions
Figure 3. Expression levels of pdc, adhA, and adhB genes in Z. mobilis
TISTR548 and Z. mobilis gfounder heat, ethanol, and osmotic stresses
after real-time RT-PCR analysis.
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Protective function of sorbitol on cell growth and ethanol
production by Z. mobilis parental and disruptant strains
Table 3. Protective function of sorbitol on cell growth and ethanol
production by Z. mobilis under heat, ethanol, and osmotic stresses
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SDS-PAGE analysis
Figure 4. SDS-PAGE analysis of protein isolated from Z. mobilis TISTR548
and Z. mobilis gfo. Growing in medium supplemented with 50 mM sorbitol
under (A) heat stress, (B) ethanol stress, and (C) osmotic stress.
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Conclusions
Sorbitol plays an important role in the process of cell
growth, ethanol fermentation, and protein synthesis not only
under osmotic stress but also under heat and ethanol stresses.
Supplementation of sorbitol into the culture medium may be
one of the effective approaches to improve the production
yield of bioethanol or other chemicals under stress conditionswith high temperature, high ethanol, or high sugar
concentrations.
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