A balanced DO‐stat and its application to the control of acetic acid excretion by recombinant <i>Escherichia coli</i>

Konstantinov, Konstantin; Kishimoto, Michimasa; Seki, Tatsuji; Yoshida, Takemi

doi:10.1002/bit.260360714

Cited by 116 publications

(56 citation statements)

References 19 publications

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“…The process parameters are summarized in Table 2. Acetic acid accumu- lated to concentrations which, accordingly to Konstantinov et al (29), do not affect cell growth in a significant way (Fig. 3B).…”

Section: Biotransformation Of M-nitrotoluene By E Coli Jm101 (Pspz3)supporting

confidence: 60%

Suitability of RecombinantEscherichia coliandPseudomonas putidaStrains for Selective Biotransformation ofm-Nitrotoluene by Xylene Monooxygenase

Meyer

Schmid

2005

Appl Environ Microbiol

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Escherichia coli JM101(pSPZ3), containing xylene monooxygenase (XMO) from Pseudomonas putida mt-2, catalyzes specific oxidations and reductions of m-nitrotoluene and derivatives thereof. In addition to reactions catalyzed by XMO, we focused on biotransformations by native enzymes of the E. coli host and their effect on overall biocatalyst performance. While m-nitrotoluene was consecutively oxygenated to m-nitrobenzyl alcohol, m-nitrobenzaldehyde, and m-nitrobenzoic acid by XMO, the oxidation was counteracted by an alcohol dehydrogenase(s) from the E. coli host, which reduced m-nitrobenzaldehyde to m-nitrobenzyl alcohol. Furthermore, the enzymatic background of the host reduced the nitro groups of the reactants resulting in the formation of aromatic amines, which were shown to effectively inhibit XMO in a reversible fashion. Host-intrinsic oxidoreductases and their reaction products had a major effect on the activity of XMO during biocatalysis of m-nitrotoluene. P. putida DOT-T1E and P. putida PpS81 were compared to E. coli JM101 as alternative hosts for XMO. These promising strains contained an additional dehydrogenase that oxidized m-nitrobenzaldehyde to the corresponding acid but catalyzed the formation of XMO-inhibiting aromatic amines at a significantly lower level than E. coli JM101.Microbial enzymes are useful catalysts for the degradation of organic pollutants in bioremediation but also for the synthesis of added-value products in biocatalytic applications. Various large-scale processes based on bacterial enzymes have been established (17,26,30,47), which illustrate the increasing impact of biocatalysis in the chemical industry (45). Heterologous gene expression has developed to a standard technology for increasing and controlling enzyme activities in bioprocesses. It allows catalysis of a variety of reactions in a limited number of bacterial strains used as recombinant hosts. The focus on a few suitable hosts is emphasized, e.g., by the PlugBug concept (DSM, Heerlen, The Netherlands). Of these strains, Escherichia coli strains are easily accessible for genetic and biochemical engineering and provide high metabolic activity for cofactor regeneration. Their use as recombinant hosts is especially favorable in cofactor-dependent oxygenase-based processes (7) for the conversion of various aromatic hydrocarbons to industrially relevant products (5, 38, 54). E. coli JM101(pSPZ3) (39) contains xylene monooxygenase (XMO) from Pseudomonas putida mt-2 and oxidizes the methyl groups of a variety of toluene and xylene derivatives to the corresponding alcohols, aldehydes, and acids (8,12,55).Nitroaromatics are widely used in large amounts as synthetic intermediates, dyes, pesticides, pharmaceuticals, and explosives (21, 46). Their electrophilic character makes them susceptible to reduction by all kinds of microbial systems (41). Numerous bacterial strains, such as those of Pseudomonas species (1, 43) and E. coli (13), provide enzymes that are able to transform nitro groups of aromatic compounds under aerobic condition...

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Section: Biotransformation Of M-nitrotoluene By E Coli Jm101 (Pspz3)supporting

confidence: 60%

Suitability of RecombinantEscherichia coliandPseudomonas putidaStrains for Selective Biotransformation ofm-Nitrotoluene by Xylene Monooxygenase

Meyer

Schmid

2005

Appl Environ Microbiol

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“…Studies indicate that the concentration at which acetate significantly reduces cell growth rate lies in the range of 3 to 5 g/liter (3,9,26,31). Although the detailed mechanism remains unknown, this byproduct is generally thought to accumulate in E. coli fermentations as a result of the tricarboxylic acid (TCA) cycle not keeping pace with glycolysis (1,24,28,36). In other words, acetate accumulates as a result of insufficient oxaloacetate being present in the first step of the TCA cycle, the conversion of oxaloacetate and acetyl-CoA to citrate via citrate synthase.…”

mentioning

confidence: 99%

Expression of an Anaplerotic Enzyme, Pyruvate Carboxylase, Improves Recombinant Protein Production in Escherichia coli

March

Eiteman

Altman

2002

Appl Environ Microbiol

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Anaplerotic enzyme reactions are those which replenish tricarboxylic acid intermediates that are withdrawn for the synthesis of biomass. In this study, we examined recombinant protein production in Escherichia coli containing activity in an additional anaplerotic enzyme, pyruvate carboxylase. In batch fermentations, the presence of pyruvate carboxylase resulted in 68% greater production of the model protein, ␤-galactosidase, 41% greater cell yield, and 57% lower acetate concentration. We discuss why these results indicate that acetate concentration does not limit cell growth and protein synthesis, as predicted by other researchers, and suggest instead that the rate of acetate formation represents an inefficient consumption of glucose carbon, which is reduced by the presence of pyruvate carboxylase.Escherichia coli is widely used for recombinant protein production, largely because it is well-characterized, fast and inexpensive to grow, and relatively easy to alter genetically (17,18). During the biochemical synthesis of proteins, production of nonessential metabolites can waste carbon and energy that might otherwise be directed toward the protein product. A prominent example of an apparently nonessential metabolite that accumulates during aerobic growth of E. coli on glucose is acetate. Enzymatically synthesized from acetyl coenzyme A (acetyl-CoA) in two steps-phosphotransacetylase (pta gene) converts acetyl-CoA to the intermediate acetyl phosphate, which is then converted to acetate with the generation of ATP by acetate kinase (ack)-acetate is also widely considered inhibitory to growth and protein production. Studies indicate that the concentration at which acetate significantly reduces cell growth rate lies in the range of 3 to 5 g/liter (3,9,26,31). Although the detailed mechanism remains unknown, this byproduct is generally thought to accumulate in E. coli fermentations as a result of the tricarboxylic acid (TCA) cycle not keeping pace with glycolysis (1,24,28,36). In other words, acetate accumulates as a result of insufficient oxaloacetate being present in the first step of the TCA cycle, the conversion of oxaloacetate and acetyl-CoA to citrate via citrate synthase.Approaches for increasing cell density or protein yield in E. coli often focus on the reduction of acetate formation, and a variety of methods have been studied. The production of acetate can be blocked altogether, for example, by mutations in the pta and/or ack genes (10,12,30,35). Alternatively, acetate accumulation can be reduced by redirecting this biochemical or its precursors to other benign biochemicals. For example, pyruvate can be converted to acetoin by acetolactate synthase (3, 4). Other methods include the following: altering glucose uptake (9), using carbon sources other than glucose (5), controlling feeding rates to better synchronize the TCA cycle and glycolysis (15, 26-29, 31, 36-38), or supplementing amino acids to reduce the demand for biosynthetic precursors (11,34). Unfortunately, many of these strategies reduce the glucose up...

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“…The pH and DO feedback feeding strategies also lowered the acetate concentration and raised the cell density in the present study of bovine E. coli fermentation. Applying the DO feedback feeding in the later fermentation period satisfies the oxygen requirements of the cells, enhancing the balance between growth rate and oxygen consumption, and inhibiting the formation of acetate [3,27] . Consequently, when the pH and DO feedback feeding strategies were combined, the cell density increased to 36.47 while the acetate accumulation decreased to 1.12 g/L.…”

Section: Discussionmentioning

confidence: 99%

Sığırlarda Escherchia coli’nin Yüksek Hücre Yoğunluğunda Fermentasyonu İçin En Uygun Kültür Koşullarının Sağlanması

Liu¹,

Li²,

Xia³

et al. 2018

Kafkas Univ Vet Fak Derg

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Bovine Escherichia coli infection, which causes major economic losses to the cattle industry each year, can be prevented by administering formalin-inactivated vaccine. However, to enhance the application of this vaccine, the cell density of the formalin-inactivated E. coli should be boosted. This can be achieved by reducing the accumulation of acetate, a primary inhibitory metabolite in E. coli fermentation. To this end, the present study investigated the effect of pH, dissolved oxygen (DO) levels, and feeding methods on bovine E. coli fermentation, and developed two-stage pH and DO control strategies and a combined pH-and DO-mediated feeding strategy for the fermentation. The optimized conditions for Bovine E. coli were pH 7.0 at 0-10 h, 6.5 at 10-24 h; DO 50% at 0-10 h, 30% at 10-24 h; pH and DO feedback feeding at 0-10 h and 10-24 h, respectively. With Bovine E. coli fermentation under the optimized conditions, the acetate accumulation was 1.12 g/L and the cell density was 36.47 (OD600), which were 59.12% lower and 77.29% higher than these with the original conditions (pH 7.0; DO 20%; residual glucose concentration maintained at 2.0 g/L). After analyzing the main nodes of acetate synthesis, it was found that the lower carbon flux enters the Embden-Meyerhof pathway. Under the optimized conditions, the pyruvate flux and acetyl-CoA synthesis were low, and much of the acetyl-CoA participated in the tricarboxylic acid cycle. The extracellular acetate flux was 8.3%, which was 65.13% lower than in the original conditions. Keywords: Escherichia coli, Acetate, Dissolved oxygen, Feeding strategy, Metabolic flux distribution Sığırlarda Escherchia coli'nin Yüksek Hücre Yoğunluğunda Fermentasyonu İçin En Uygun Kültür Koşullarının Sağlanması ÖzSığırlardaki Escherchia coli enfeksiyonları her yıl sığırcılık sektörünü büyük çaplı maddi zarara uğratmakla birlikte, formalinle etkisizleştirilmiş bakteriden yapılan aşıyla önlenebilmektedir. Ancak, bu aşının etkisini artırmak için formalinde etkisizleştirmiş E. coli hücre yoğunluğunun artırılması gerekmektedir. Bunu elde etmek için E. coli fermentasyonunu baskılayan birincil bir metabolik ürün olan asetatın birikimi azaltılmalıdır. Bu amaçla, mevcut çalışmada pH, çözünmüş oksijen (ÇO) düzeyleri ve beslenme şeklinin sığırlardaki E. coli fermentasyonuna etkileri araştırılmış ve iki aşamalı bir pH ve ÇO kontrolü stratejisi geliştirilerek pH ve ÇO düzeylerini birlikte değerlendiren bir beslenme planı ile fermentasyonun artırılması yoluna gidilmiştir. Sığır E. coli için optimize edimiş koşullar pH için 0-10 saat aralığında 7.0, 10-24 saat aralığında 6.5; ÇO için 0-10 saat aralığında %50, 10-24 saat aralığında %30, pH ve ÇO temelinde besleme için sırasıyla 0-10 saat ve 10-24 saat olarak belirlendi. Optimize koşullar altında sığır E. coli fermentasyonu ile birlikte asetat birikimi 1.12 g/L olup hücre yoğunluğu 36.47 (OD600) olarak tespit edildi. Bu değerler orjinal koşullardan (pH 7.0; ÇO %20; kalıntı glukoz derişimi 2.0 g/L'de tutulmuş) sırasıyla %59.12 daha düşük ve %77.29 daha...

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A balanced DO‐stat and its application to the control of acetic acid excretion by recombinant Escherichia coli

Cited by 116 publications

References 19 publications

Suitability of RecombinantEscherichia coliandPseudomonas putidaStrains for Selective Biotransformation ofm-Nitrotoluene by Xylene Monooxygenase

Suitability of RecombinantEscherichia coliandPseudomonas putidaStrains for Selective Biotransformation ofm-Nitrotoluene by Xylene Monooxygenase

Expression of an Anaplerotic Enzyme, Pyruvate Carboxylase, Improves Recombinant Protein Production in Escherichia coli

Sığırlarda Escherchia coli’nin Yüksek Hücre Yoğunluğunda Fermentasyonu İçin En Uygun Kültür Koşullarının Sağlanması

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