Direct conversion of glucose to malate by synthetic metabolic engineering

Ye, Xiaoting; Honda, Kohsuke; Morimoto, Yumi; Okano, Kenji; Ohtake, Hisao

doi:10.1016/j.jbiotec.2012.11.011

Cited by 54 publications

(41 citation statements)

References 28 publications

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“…The expression vector for the Thermus thermophilus fumarase (TtFTA) was obtained from the RIKEN Thermus thermophilus HB8 expression plasmid library (14) and designated pET-TtFTA. The expression vector for the malic enzyme of Thermococcus kodakarensis KOD1 (TkME) was constructed as described elsewhere (15). E. coli Rosetta 2 (DE3) pLysS (Novagen, Madison, WI) was used as the host cell for gene expression.…”

Section: Methodsmentioning

confidence: 99%

Development of a Continuous Bioconversion System Using a Thermophilic Whole-Cell Biocatalyst

Ninh

Honda

Yokohigashi

et al. 2013

Appl Environ Microbiol

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The heat treatment of recombinant mesophilic cells having heterologous thermophilic enzymes results in the denaturation of indigenous mesophilic enzymes and the elimination of undesired side reactions; therefore, highly selective whole-cell catalysts comparable to purified enzymes can be readily prepared. However, the thermolysis of host cells leads to the heat-induced leakage of thermophilic enzymes, which are produced as soluble proteins, limiting the exploitation of their excellent stability in repeated and continuous reactions. In this study, Escherichia coli cells having the thermophilic fumarase from Thermus thermophilus (TtFTA) were treated with glutaraldehyde to prevent the heat-induced leakage of the enzyme, and the resulting cells were used as a whole-cell catalyst in repeated and continuous reactions. Interestingly, although electron microscopic observations revealed that the cellular structure of glutaraldehyde-treated E. coli was not apparently changed by the heat treatment, the membrane permeability of the heated cells to relatively small molecules (up to at least 3 kDa) was significantly improved. By applying the glutaraldehyde-treated E. coli having TtFTA to a continuous reactor equipped with a cell-separation membrane filter, the enzymatic hydration of fumarate to malate could be operated for more than 600 min with a molar conversion yield of 60% or higher.T hermophilic enzymes are promising tools for biotransformation owing to their high operational stability, cosolvent compatibility, and low risk of contamination (1-3). The direct use of recombinant mesophiles (e.g., Escherichia coli) having heterologous thermophilic enzymes at high temperatures results in the denaturation of indigenous enzymes and the elimination of undesired side reactions; therefore, highly selective whole-cell catalysts comparable to purified enzymes can be readily prepared. Honda et al. have demonstrated that the rational combination of these thermophilic whole-cell catalysts enables the construction of in vitro artificial biosynthetic pathways for the production of value-added chemicals (4). Recently, Ye et al. have successfully constructed a chimeric Embden-Meyerhof pathway with the balanced consumption and regeneration of ATP and ADP, using nine recombinant E. coli strains, each of which overproduces a thermophilic glycolytic enzyme (5).The membrane structure of E. coli cells is partially or entirely disrupted at high temperatures, and thus thermophilic enzymes, which are produced as soluble proteins, leak out of the cells (6-9). Although the heat-induced leakage of thermophilic enzymes results in better accessibility between the enzymes and substrates, it limits the applicability of thermophilic whole-cell catalysts to continuous and repeated-batch reaction systems. This limitation prevents us from exploiting the most advantageous feature of thermophilic biocatalysts, namely, their excellent stability. To overcome this limitation, one potential strategy is the integration of thermophilic enzymes to the membrane struct...

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Section: Methodsmentioning

confidence: 99%

Development of a Continuous Bioconversion System Using a Thermophilic Whole-Cell Biocatalyst

Ninh

Honda

Yokohigashi

et al. 2013

Appl Environ Microbiol

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“…Psychrophile-based simple biocatalysts may be able to exploit nearly all enzymes to produce chemicals. Furthermore, decreasing the heat treatment temperature to an ordinary temperature limits concerns about the excess disruption of the cellular structure and thermal decomposition of coenzymes, such as NADP + and NADPH, which are problems in E. coli -based simple biocatalysts (Ye et al 2013). …”

Section: Introductionmentioning

confidence: 99%

Construction of a simple biocatalyst using psychrophilic bacterial cells and its application for efficient 3-hydroxypropionaldehyde production from glycerol

et al. 2013

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Most whole cell biocatalysts have some problems with yields and productivities because of various metabolites produced as byproducts and limitations of substrate uptake. We propose a psychrophile-based simple biocatalyst for efficient bio-production using mesophilic enzymes expressed in psychrophilic Shewanella livingstonensis Ac10 cells whose basic metabolism was inactivated by heat treatment. The 45°C heat-treated cells expressing lacZ showed maximum beta-galactosidase activity as well as chloroform/SDS-treated cells to increase membrane permeability. The fluorescent dye 5-cyano-2,3-ditolyl-tetrazolium chloride staining indicated that most basic metabolism of Ac10 was lost by heat treatment at 45˚C for 10 min. The simple biocatalyst was applied for 3-HPA production by using Klebsiella pneumoniae dhaB genes. 3-HPA was stoichiometrically produced with the complete consumption of glycerol at a high production rate of 8.85 mmol 3-HPA/g dry cell/h. The amount of 3-HPA production increased by increasing the concentrations of biocatalyst and glycerol. Furthermore, it could convert biodiesel-derived crude glycerol to 3-HPA.

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“…Traditionally, in vitro pathway construction has been relegated to use as a research tool or in applications that require only 1-3 enzymes for the production of chiral compounds and other highvalue chemicals 1,8,9 . Improvements in protein expression and access to stable enzymes have made more complex systems possible, however 1,[9][10][11][12] .…”

mentioning

confidence: 99%

“…Improvements in protein expression and access to stable enzymes have made more complex systems possible, however 1,[9][10][11][12] . In vitro biotransformation systems have been reported in recent years involving systems of over 30 enzymes 13,14 .…”

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confidence: 99%

A synthetic biochemistry molecular purge valve module that maintains redox balance

2014

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Direct conversion of glucose to malate by synthetic metabolic engineering

Cited by 54 publications

References 28 publications

Development of a Continuous Bioconversion System Using a Thermophilic Whole-Cell Biocatalyst

Development of a Continuous Bioconversion System Using a Thermophilic Whole-Cell Biocatalyst

Construction of a simple biocatalyst using psychrophilic bacterial cells and its application for efficient 3-hydroxypropionaldehyde production from glycerol

A synthetic biochemistry molecular purge valve module that maintains redox balance

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