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

Abstract

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...

Production of phenylpyruvic acid from l-phenylalanine using an l-amino acid deaminase from Proteus mirabilis: comparison of enzymatic and whole-cell biotransformation approaches

“…These results suggest that, compared with the pure enzyme, the whole-cell catalyst has better thermal stability during the transformation of L-phenylalanine to PPA. Many other whole-cell catalysts also showed good thermal stability, like the recombinant E. coli whole-cell catalyst co-expressing glycerol dehydrogenase and glucose dehydrogenase (Richter et al 2010), and the E. coli cells expressing the thermophilic fumarase from Thermus thermophilus (Ninh et al 2013).…”

Recent advances in whole cell biocatalysis techniques bridging from investigative to industrial scale

“…Applications in which immobilization was used to enable highly productive reactor concepts were published recently with glutaraldehyde-crosslinked whole cell catalyst [55,56]. In the example given by Stojkovič and Ž nidaršič-Plazl a 25 mL microreactor could be loaded with such a high catalyst load that space-time-yields 3.5 times higher than in a comparable 1 L membrane reactor were achieved [56].…”

Cell‐free metabolic engineering: Biomanufacturing beyond the cell

“…In an effort to improve stability of the thermophilic enzymes themselves, glutaraldehyde was used to crosslink proteins so that the E. coli chassis cell remained intact after heat treatment [87]. The membrane in this state is permeable to small molecules and the “whole cells” could repeatedly convert fumarate to malate.…”