In vitro Metabolic Engineering

Bioproduction of value-added chemicals has been widely recognized as a key technology to promote the sustainability of chemical industries. Our group has developed a novel technology to engineer an artificial metabolic pathway specialized for chemical manufacturing by assembling multiple enzymes in vitro. Enzymes from thermophilic microorganisms are heterologously expressed in mesophilic hosts (e.g., Escherichia coli) and used as catalytic modules for pathway construction. This approach is, in principle, available for all thermophilic enzymes as long as they can be functionally expressed in mesophilic hosts, allowing us to design a bespoke metabolic pathway in vitro. We referred to this technology as “in vitro metabolic engineering” and construct a variety of artificial pathways for the on-demand production of useful chemicals.

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Non-aqueous Bioprocess

The use of organic solvents as reaction media has the potential to greatly expand the repertoire of bioproduction. Successful examples of bioproduction in nonaqueous media using enzymes including lyophilized lipases, esterases, and proteases have been reported. However, most of these biocatalytic convesions involve relatively simple, single-step hydrolytic enzymes. One of the challenges in this area is the utilization of more complex enzymatic reactions involving multicomponent enzymes, cofactor regeneration, and multistep enzymatic conversions in organic solvent. Theerfore, attention has been paid to the development of a whole-cell catalyst that functions in nonaqueous environment. Rhodococcus opacus B-4 cells exhibit a high affinity for water-immiscible hydrocarbons and dispersible in essentially water-free organic solvents. Owing to these hydrophobic features, R. opacus B-4 can easily access to the water-insoluble chemicals dissolved in organic solvnets and have an advantage in taking up them. By using R.opacus B-4 cells as "hydrophobic biocatalytic particles", we are developing nonaquesous bioprocesses for the production of water-insoluble chemicals.


Behavior of Rhodococcus opacus and Escherichia coli in a water-immiscible organic solvent.
The wet cells of R. oapcus (left) and E. coli (right) were put in an Erlenmeyer flask containing cyclohexane and mixed with a magnetic stirrer. Although E. coli cells are repelled from the solvent, R. opacus cells can be suspended in cyclohexane.

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