A two-step biocatalytic cascade in micro-aqueous medium: using whole cells to obtain high concentrations of a vicinal diol

Although single-and multi-step biocatalytic approaches show persuasive advantages for the synthesis of especially chiral compounds (e.g. high chemo-and stereoselectivity), their application often suffers from low substrate loads and hence low space-time-yields. We herein present a synthetic cascade approach in which lyophilised, recombinant whole cells are applied in micro-aqueous reaction systems yielding extremely high space-time-yields. As an example we investigated the two-step synthesis of 1-phenylpropane-1,2-diol starting from cheap aldehydes and achieved high selectivities (ee/de > 99%) and high product concentrations. The new concept of running biocatalytic cascades in a mixture of high substrate loads and organic solvents under addition of small amounts of highly concentrated buffer is not only very easy-to-apply, but also exhibits several economic and ecologic advantages. On the one hand the usage of whole, lyophilised cells circumvents time-consuming enzyme purification as well as addition of expensive cofactors (here ThDP and NADPH). Additionally, catalyst and product workup is facilitated by the application of organic solvents (here MTBE). On the other hand, the employment of whole cells very effectively circumvents stability problems of biocatalysts in unconventional media enabling the addition of extremely high substrate loads (up to 500 mM in our example) and is therefore an easy and effective approach for multi-step biocatalysis. After optimisation, the combination of a carboligation step followed by a second oxidoreduction step with whole cell catalysts afforded an efficient two-step cascade for the production of 1-phenylpropane-1,2-diol with space-time yields up to 327 g L-1 d(-1) and an E-factor of 21.3 kg(waste) kg(product)(-1).