In vitro reconstitution of an artificial metabolic pathway is an emerging approach for the biocatalytic production of industrial chemicals. However, several enzymes have to be separately prepared (and purified) for the construction of an in vitro metabolic pathway, thereby limiting the practical applicability of this approach. In this study, genes encoding the nine thermophilic enzymes involved in a non-ATP-forming chimeric glycolytic pathway were assembled in an artificial operon and co-expressed in a single recombinant Escherichia coli strain. Gene expression levels of the thermophilic enzymes were controlled by their sequential order in the artificial operon. The specific activities of the recombinant enzymes in the cell-free extract of the multiple-gene-expression E. coli were 5.0-1,370 times higher than those in an enzyme cocktail prepared from a mixture of single-gene-expression strains, in each of which a single one of the nine thermophilic enzymes was overproduced. Heat treatment of a crude extract of the multiple-gene-expression cells led to the denaturation of indigenous proteins and one-step preparation of an in vitro synthetic pathway comprising only a limited number of thermotolerant enzymes. Coupling this in vitro pathway with other thermophilic enzymes including the H2 O-forming NADH oxidase or the malate/lactate dehydrogenase facilitated one-pot conversion of glucose to pyruvate or lactate, respectively.

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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