Betaine Tripled the Volumetric Productivity of d(-)-lactate by Escherichia coli Strain SZ132 in Mineral Salts Medium

Zhou, Shengde; Grabar, Tammy Bohannon; Shanmugam, K. T.; Ingram, L. O.

doi:10.1007/s10529-006-0033-4

Cited by 52 publications

(45 citation statements)

References 15 publications

(16 reference statements)

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“…Zhou et al (2006) reported that d-())-lactate synthesis significantly increased when betaine was added to the culture medium as an osmoprotectant. It was likewise found that modification of the trehalose content in recombinant E. coli also resulted in higher growth (Purvis et al 2005), and trehalose-dependent stress tolerance was identified as a relevant trait for industrial purposes involving baker's yeast (Attfield 1997).…”

Section: Discussionmentioning

confidence: 99%

Ethanol synthesis from glycerol by Escherichia coli redox mutants expressing adhE from Leuconostoc mesenteroides

Nikel

Ramírez

Pettinari

et al. 2010

Journal of Applied Microbiology

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Aims: Analysis of the physiology and metabolism of Escherichia coli arcA and creC mutants expressing a bifunctional alcohol‐acetaldehyde dehydrogenase from Leuconostoc mesenteroides growing on glycerol under oxygen‐restricted conditions. The effect of an ldhA mutation and different growth medium modifications was also assessed. Methods and Results: Expression of adhE in E. coli CT1061 [arcA creC(Con)] resulted in a 1·4‐fold enhancement in ethanol synthesis. Significant amounts of lactate were produced during micro‐oxic cultures and strain CT1061LE, in which fermentative lactate dehydrogenase was deleted, produced up to 6·5 ± 0·3 g l−1 ethanol in 48 h. Escherichia coli CT1061LE derivatives resistant to >25 g l−1 ethanol were obtained by metabolic evolution. Pyruvate and acetaldehyde addition significantly increased both biomass and ethanol concentrations, probably by overcoming acetyl‐coenzyme A (CoA) shortage. Yeast extract also promoted growth and ethanol synthesis, and this positive effect was mainly attributable to its vitamin content. Two‐stage bioreactor cultures were conducted in a minimal medium containing 100 μg l−1 calcium d‐pantothenate to evaluate oxic acetyl‐CoA synthesis followed by a switch into fermentative conditions. Ethanol reached 15·4 ± 0·9 g l−1 with a volumetric productivity of 0·34 ± 0·02 g l−1 h−1. Conclusions: Escherichia coli responded to adhE over‐expression by funnelling carbon and reducing equivalents into a highly reduced metabolite, ethanol. Acetyl‐CoA played a key role in micro‐oxic ethanol synthesis and growth. Significance and Impact of the Study: Insight into the micro‐oxic metabolism of E. coli growing on glycerol is essential for the development of efficient industrial processes for reduced biochemicals production from this substrate, with special relevance to biofuels synthesis.

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Section: Discussionmentioning

confidence: 99%

Ethanol synthesis from glycerol by Escherichia coli redox mutants expressing adhE from Leuconostoc mesenteroides

Nikel

Ramírez

Pettinari

et al. 2010

Journal of Applied Microbiology

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“…All controlled batch bioreactor studies were performed with defined minimal medium containing 3.5 g/liter of KH 2 PO 4 , 5.0 g/liter of K 2 HPO 4 , 3.5 g/liter of (NH 4 ) 2 HPO 4 , 0.25 g/liter of MgSO 4 ⅐ 7H 2 O, 15 mg/ liter of CaCl 2 ⅐ 2H 2 O, 0.5 mg/liter of thiamine, 0.12 g/liter of betaine, 1 ml/liter of a stock trace metal solution, 40 g/liter of glycerol (unless otherwise specified), and 10 g/liter of tetracycline (adapted from media described previously [6,21,26] 4 ) citrate. The first three salt components of the defined minimal medium were autoclaved, while the other components were sterile filtered and added to bioreactors.…”

Section: Methodsmentioning

confidence: 99%

Metabolic Engineering of Escherichia coli for Efficient Conversion of Glycerol to Ethanol

Trinh

Srienc

2009

Appl Environ Microbiol

135

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Based on elementary mode analysis, an Escherichia coli strain was designed for efficient conversion of glycerol to ethanol. By using nine gene knockout mutations, the functional space of the central metabolism of E. coli was reduced from over 15,000 possible pathways to a total of 28 glycerol-utilizing pathways that support cell function. Among these pathways are eight aerobic and eight anaerobic pathways that do not support cell growth but convert glycerol into ethanol with a theoretical yield of 0.50 g ethanol/g glycerol. The remaining 12 pathways aerobically coproduce biomass and ethanol from glycerol. The optimal ethanol production depends on the oxygen availability that regulates the two competing pathways for biomass and ethanol production. The coupling between cell growth and ethanol production enabled metabolic evolution of the designed strain through serial dilution that resulted in strains with improved ethanol yields and productivities. In defined medium, the evolved strain can convert 40 g/liter of glycerol to ethanol in 48 h with 90% of the theoretical ethanol yield. The performance of the designed strain is predicted by the property space of remaining elementary modes.

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“…Because the accumulation of pyruvate in our current study would simultaneously require the accumulation of K ϩ (or another counterion) to maintain the pH, we conducted an experiment to determine whether the addition of the known osmoprotectant betaine (38,50) would improve pyruvate accumulation. Without betaine present in the medium under acetate-limited conditions, strain ALS929 was able to accumulate over 56 g/liter pyruvate in about 28 h (Fig.…”

Section: Growth-limiting Nutrientmentioning

confidence: 99%

“…Another explanation for the observed termination of cell growth and pyruvate formation is the high osmotic stress caused by the K ϩ ion required to maintain the pH. Organic osmolytes, such as betaine, often accumulate naturally to protect cells from high osmotic stress (44), and the production of ethanol (38) and lactate (50) has been improved by the addition of betaine to the medium. Our fed-batch results similarly demonstrated that 5 mM betaine prolonged cell growth and thereby increased the final pyruvate concentration by at least 25%.…”

Section: Vol 74 2008mentioning

confidence: 99%

High Glycolytic Flux Improves Pyruvate Production by a Metabolically Engineered Escherichia coli Strain

Zhu

Eiteman

Altman

et al. 2008

Appl Environ Microbiol

104

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We report pyruvate formation in Escherichia coli strain ALS929 containing mutations in the aceEF, pfl, poxB, pps, and ldhA genes which encode, respectively, the pyruvate dehydrogenase complex, pyruvate formate lyase, pyruvate oxidase, phosphoenolpyruvate synthase, and lactate dehydrogenase. The glycolytic rate and pyruvate productivity were compared using glucose-, acetate-, nitrogen-, or phosphorus-limited chemostats at a growth rate of 0.15 h ؊1 . Of these four nutrient limitation conditions, growth under acetate limitation resulted in the highest glycolytic flux (1.60 g/g · h), pyruvate formation rate (1.11 g/g · h), and pyruvate yield (0.70 g/g). Additional mutations in atpFH and arcA (strain ALS1059) further elevated the steady-state glycolytic flux to 2.38 g/g · h in an acetate-limited chemostat, with heterologous NADH oxidase expression causing only modest additional improvement. A fed-batch process with strain ALS1059 using defined medium with 5 mM betaine as osmoprotectant and an exponential feeding rate of 0.15 h ؊1 achieved 90 g/liter pyruvate, with an overall productivity of 2.1 g/liter · h and yield of 0.68 g/g.Pyruvic acid (pyruvate) is widely used in food, chemicals, and pharmaceuticals. The chemical is a precursor for the enzymatic production of L-tryptophan, L-tyrosine, D-/L-alanine, and L-dihydroxyphenylalanine (22), and it also serves in several health-related roles, including weight loss (25,33,34), exercise endurance (32), cholesterol reduction (35), and acne treatment (10). Recently, pyruvate has been used as the key metabolic precursor to the second-generation biofuels isobutanol and 3-methyl-1-butanol (2). By applying metabolic engineering strategies to them, microorganisms such as Escherichia coli and yeasts can be used to produce significant quantities of pyruvate from glucose and other renewable resources (22). In general, such approaches must delete or repress pathways which metabolize pyruvate. For example, pyruvate accumulates readily in E. coli strains having mutations in aceEF, encoding components of the pyruvate dehydrogenase complex (37). Additional mutations, of the ldhA, poxB, pfl, and pps genes, further improve pyruvate formation (48). Figure 1 shows the principal metabolic pathways and enzymes involved in the formation of pyruvate.Because pyruvate resides biochemically at the end of glycolysis, pyruvate production is directly related to the glycolytic flux. Metabolic engineering strategies to form pyruvate therefore also aim to enhance glycolysis (8,45,46). Glycolysis is not transcriptionally limited, and control principally resides outside the pathway in cellular demand for global cofactors, such as ATP and NADH (19,23,40). Glycolytic flux is substantially increased by disrupting oxidative phosphorylation or by increasing ATP hydrolysis (8,19). Increased glycolytic flux assists pyruvate accumulation: for example, an F 1 -ATPase-defective mutant (E. coli lipA2 bgl ϩ atpA401) generated pyruvate more quickly than its parent (45,46). Similarly, E. coli atpFH (strain TC44), defi...

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Betaine Tripled the Volumetric Productivity of d(-)-lactate by Escherichia coli Strain SZ132 in Mineral Salts Medium

Cited by 52 publications

References 15 publications

Ethanol synthesis from glycerol by Escherichia coli redox mutants expressing adhE from Leuconostoc mesenteroides

Ethanol synthesis from glycerol by Escherichia coli redox mutants expressing adhE from Leuconostoc mesenteroides

Metabolic Engineering of Escherichia coli for Efficient Conversion of Glycerol to Ethanol

High Glycolytic Flux Improves Pyruvate Production by a Metabolically Engineered Escherichia coli Strain

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