Comparison of growth, acetate production, and acetate inhibition of Escherichia coli strains in batch and fed-batch fermentations

Luli, G W; Strohl, William R.

doi:10.1128/aem.56.4.1004-1011.1990

Cited by 525 publications

(270 citation statements)

References 20 publications

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“…For both cases the cell will attempt to balance carbon fluxes that result mainly with acetate as the major byproduct (Holms, 1986). Acetate, is a lipophilic agent that is harmful to cell growth (Adams, 1988;Luli and Strohl, 1990). Moreover, experimental results in our laboratory (Chou et al, 1995) agree well with common observation that recombinant gene expression is greatly reduced for acetate accumulation above 15-25 mM (Bauer et al, 1990;Jensen and Carlsen, 1990;Shimuzu et al, 1988).…”

Section: Introductionsupporting

confidence: 86%

Metabolic Engineering of Escherichia coli To Enhance Recombinant Protein Production through Acetate Reduction

Aristidou

San

Bennett

1995

Biotechnology Progress

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Genetic and metabolic engineering provide powerful and effective tools for the systematic manipulation and fine tuning of cellular metabolic activities. In this study, successful application of such techniques to enhance recombinant protein production by reducing acetate accumulation in Escherichia coli is presented. The alsS gene from Bacillus subtilis encoding the enzyme acetolactate synthase was introduced into E. coli cells using a multicopy plasmid. This newly introduced heterologous enzyme modifies the glycolytic fluxes by redirecting excess pyruvate away from acetate to acetolactate. Acetolactate is then converted to a nonacidic and less harmful byproduct acetoin, which appears in the broth. Furthermore, comparative fermentation studies show that the reduction in acetate accumulation leads to a significant improvement of recombinant protein production. The expression of a model recombinant CadA/beta-galactosidase fusion protein, under the control of a strong pH-regulated promoter, was found to increase by about 60% for the specific protein activity (to a level of 30% of total cellular protein) and 50% in terms of the volumetric activity in a batch fermenter. In fed-batch cultivation, the engineered strain achieved a volumetric recombinant protein yield of 1.6 million units/mL (about 1.1 g/L of beta-galactosidase), which represented a 220% enhancement over the control strain. In the meantime, acetate excretion was maintained below 20 mM compared with 80 mM for the control, and the final cell density was improved by 35%.

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Section: Introductionsupporting

confidence: 86%

Metabolic Engineering of Escherichia coli To Enhance Recombinant Protein Production through Acetate Reduction

Aristidou

San

Bennett

1995

Biotechnology Progress

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“…When the cells are not under starvation, they uptake excess glucose beyond that utilized for biomass synthesis and therefore excrete the carbon source in the form of acetate (Doelle et al, 1981;Farmer and Liao, 1997;Kleman and Strohl, 1994). In addition to a loss of carbon and therefore an economic sink, acetate is also detrimental to recombinant protein production because it inhibits cell growth (Hahm et al, 1994;Holms, 1986;Luli and Strohl, 1990). While acetate production depends on many factors including the growth medium, glucose feeding strategy, growth conditions, and bacterial strain (van de Walle and Shiloach, 1998), it remains a universal problem when using E. coli.…”

Section: Introductionmentioning

confidence: 99%

Characterization of the Acetate-Producing Pathways in Escherichia coli

Dittrich

Bennett

San

2008

Biotechnol Progress

116

100

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Although the bacterium E. coli is chosen as the host in many bioprocesses, the accumulation of a common byproduct, acetate, is often problematic. Acetate, when present at high levels, will inhibit both cell growth and recombinant protein productivity. In addition, products derived from the central aerobic metabolic pathway often compete with the acetate-producing pathways poxB and ackA-pta for glucose as the substrate. As such, a significant portion of the glucose may be excreted as acetate, wasting substrate that otherwise could have been used for the desired product. We have created mutant E. coli strains with a deletion of either the poxB or the ackA-pta pathway. These two strains, along with the wild-type strain, have been studied in batch reactors over a 12 h time period, at pH 7.0 and 6.0. The wild-type strain has also been studied using glucose as the carbon source. Data were collected to correlate cellular growth, extracellular metabolite production, enzyme activity, and gene expression. Results show that the ackA-pta pathway dominates in exponential phase, and the poxB pathway dominates in stationary phase. The ackA-pta pathway is repressed in acidic environments, whereas the poxB pathway is activated.

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“…Among the many factors influencing the growth of recombinant E. coli, acetate or acetate-producing environment is known to affect the cell growth and gene expression. In the cultivation of recombinant E. coli, an acetate concentration above 2 g/L was known to inhibit cell growth (Luli and Strohl, 1990). As shown in Table 2, when acetate formation was compared between glucose and glycerol in a flask culture, glycerol gave one-third of the acetate produced from glucose.…”

Section: Resultsmentioning

confidence: 99%

“…In order to cultivate cells at high density, feed-on-demand fed-batch techniques, such as DO-stat or pH-stat, can be employed (O'Conner et al, 1992). During the cultivation of recombinant E. coli, accumulation of acetate in the medium decreases the growth rate as well as gene expression (Luli and Strohl, 1990). Therefore most feeding strategies are designed to prevent acetate formation by various methods including the maintenance of the glucose concentration as low as possible.…”

Section: Introductionmentioning

confidence: 99%

Effects of Glycerol on β‐Lactamase Production during High Cell Density Cultivation of Recombinant Escherichia coli

Kwon

Kim

1996

Biotechnology Progress

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Recombinant Escherichia coli was cultured up to OD600 90 with glycerol fed by the DO-STAT method to produce recombinant beta-lactamase. Optimum feeding and induction conditions were determined. When glycerol was maintained at a high concentration (40-50 g/L) after isopropyl thiogalactoside (IPTG) induction at OD600 75, a 2-fold increase in the amount of soluble beta-lactamase was obtained. Similarly, a 3-fold increase was obtained by the addition of NaCl (0.4 M) at low glycerol concentration (10 g/L).

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Comparison of growth, acetate production, and acetate inhibition of Escherichia coli strains in batch and fed-batch fermentations

Cited by 525 publications

References 20 publications

Metabolic Engineering of Escherichia coli To Enhance Recombinant Protein Production through Acetate Reduction

Metabolic Engineering of Escherichia coli To Enhance Recombinant Protein Production through Acetate Reduction

Characterization of the Acetate-Producing Pathways in Escherichia coli

Effects of Glycerol on β‐Lactamase Production during High Cell Density Cultivation of Recombinant Escherichia coli

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