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 easyto-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 . † Electronic supplementary information (ESI) available. See Scheme 1 Model two-step reaction cascade composed of asymmetric carboligation by BAL (step 1) and subsequent ketone reduction by RADH (step 2). BAL = benzaldehyde lyase from Pseudomonas fluorescens, RADH = alcohol dehydrogenase from Ralstonia sp., (2R)-HPP = 2-hydroxy-1phenylpropan-1-one, PPD = 1-phenylpropane-1,2-diol. Green Chemistry PaperThis journal is

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Combining enzymes to form multi‐step enzyme cascades has great potential to replace existing chemical routes with high atom‐efficient and eco‐efficient synthesis strategies as well as to grant access to new products, especially those with multi‐stereogenic centres. However, easy solutions and tools for setting up appropriate reaction conditions and process modes are hardly available. The utilisation of teabags filled with whole cells has several advantages, such as 1) simplified handling and recovery of catalyst, 2) easy combination of various catalysts from catalyst toolboxes, 3) fast testing of different operating modes during cascadation and 4) simplified downstream processing. One of the main advantages is that lyophilised whole‐cell catalysts can be applied in micro‐aqueous media, allowing high substrate loads (also of poorly water‐soluble substrates) and concomitantly enabling high catalyst stability. This was demonstrated herein for a synthetic two‐step cascade towards chiral 1,2‐diols starting from cheap aldehydes. The carboligation of two aldehydes using Pseudomonas fluorescens benzaldehyde lyase and subsequent oxidoreduction with Ralstonia sp. alcohol dehydrogenase yielded 1‐phenylpropane‐1,2‐diol [(1R,2R)‐PPD] in concentrations of up to 339 mM and excellent enantiomeric and diastereomeric excesses >99 %. Therefore, the combination of whole‐cell catalysis and teabag modularisation allows cheap, easy‐to‐apply and efficient catalyst preparation to test enzyme combinations and optimal reaction conditions up to the preparative scale. By circumventing catalyst purification and immobilisation, and enabling high substrate loadings compared to those in aqueous systems, efficient production of a chiral diol with extraordinarily high product concentrations can be achieved.

Biotransformations on larger scale are mostly limited to cases in which alternative chemical routes lack sufficient chemo-, regio-, or stereoselectivity. Here, we expand the applicability of biocatalysis by combining cheap whole cell catalysts with a microaqueous solvent system. Compared to aqueous systems, this permits manifoldly higher concentrations of hydrophobic substrates while maintaining stereoselectivity. We apply these methods to four different two-step reactions of carboligation and oxidoreduction to obtain 1-phenylpropane-1,2-diol (PPD), a versatile building block for pharmaceuticals, starting from inexpensive aldehyde substrates. By a modular combination of two carboligases and two alcohol dehydrogenases, all four stereoisomers of PPD can be produced in a flexible way. After thorough optimization of each two-step reaction, the resulting processes enabled up to 63 g L −1 product concentration (98% yield), space-time-yields up to 144 g L −1 d −1 , and a target isomer content of at least 95%. Despite the use of whole cell catalysts, we did not observe any side product formation of note. In addition, we prove that, by using 1,5-pentandiol as a smart cosubstrate, a very advantageous cofactor regeneration system could be applied.

The carbonyl reductase from Candida parapsilosis (CPCR2) is a versatile biocatalyst for the production of optically pure alcohols from ketones. Prochiral ketones like 2-methyl cyclohexanone are, however, only poorly accepted, despite CPCR2's large substrate spectrum. The substrate spectrum of CPCR2 was investigated by selecting five amino positions (55, 92, 118, 119 and 262) and exploring them by single site-saturation mutagenesis. Screening of CPCR2 libraries with poor (14 compounds) and well-accepted (2 compounds) substrates showed that only position 55 and position 119 showed an influence on activity. Saturation of positions 92, 118 and 262 yielded only wild-type sequences for the two well-accepted substrates and no variant converted one of the 14 other compounds. Only the variant (L119M) showed a significantly improved activity (7-fold on 2-methyl cyclohexanone; vmax = 33.6 U/mg, Km = 9.7 mmol/l). The L119M substitution exhibited also significantly increased activity toward reduction of 3-methyl (>2-fold), 4-methyl (>5-fold) and non-substituted cyclohexanone (>4-fold). After docking 2-methyl cyclohexanone into the substrate-binding pocket of a CPCR2 homology model, we hypothesized that the flexible side chain of M119 provides more space for 2-methyl cyclohexanone than branched L119. This report represents the first study on CPCR2 engineering and provides first insights how to redesign CPCR2 toward a broadened substrate spectrum.

Proof of identity: The previously described ability of Candida parapsilosis lysate to reduce structurally diverse ketones led to the discovery of two carbonyl reductases as possible candidates for this interesting biotransformation. Homology modelling and prediction of indicator substrates helped to identify which enzyme has the large substrate scope verified by biochemical data.

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