sorbitol required for cell growth and ethanol production

8/10/2019 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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sorbitol required for cell growth and ethanol production

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