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

Abstract

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

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

“…Alternatively, water or buffer is added resulting in a micro-aqueous system, in which ideally no second phase is observed as the water is taken up by the solvent or the catalyst [27]. In 2011, Jakoblinnert et al applied lyophilized E. coli whole cells expressing Candida parapsilosis carbonyl reductase (CPCR) in a mixture of only acetophenone as main substrate and n-propanol as a cosubstrate for cofactor regeneration.…”

“…Nevertheless, substrate concentrations of 500 mM could be achieved in micro-aqueous MTBE in a 2-step reaction employing recombinant whole cell catalyst for carboligation and oxidoreduction, respectively. Even with highly inactivating acetaldehyde and benzaldehyde as substrates, space-time-yields and product concentration of the batch reactions were outstandingly high, with up to 327 g L À1 d À1 and 49 g L À1 for (1R,2R)-phenylpropane-1,2-diol production [27].…”

“…On the other hand, coexpression of multiple proteins may lead to a high metabolic burden during cell growth, which can result in poor overexpression and thus impaired catalytic performance [46]. Therefore, in some cases it can be beneficial to express each enzyme in single and later combine the cells into cascade reactions flexibly [27]. Anyhow, cascade biocatalysis becomes particularly sophisticated yet appealing when redox-neutral (see Section 'Cofactor regeneration') or recycling cascades [47] are designed.…”

Biocatalytic Racemization Employing TeSADH: Substrate Scope and Organic Solvent Compatibility for Dynamic Kinetic Resolution

“…Therefore, we decided to test E. coli /TeSADH WIC in an organic solvent, especially since both the wild‐type enzyme and the W110A have already been reported to be active in organic solvents . Inspired by the results of reactions catalyzed by lyophilized ADHs in micro‐aqueous systems (μAq), we decided to adapt similar reaction conditions and test whether the E. coli /TeSADH WIC would be active and still maintain low enantioselectivity. Oxidation of rac ‐ 1 b to 2 b under μAq conditions indicated E. coli /TeSADH WIC retains activity and low enantioselectivity in selected solvents, such as n ‐hexane, cyclohexane, toluene, diisopropyl ether (DIPE), methyl tert ‐butyl ether (MTBE), or ethyl acetate (Table , entries 1–6).…”

Highly Efficient Synthesis of Optically Pure (S)‐1‐phenyl‐1,2‐ethanediol by a Self‐Sufficient Whole Cell Biocatalyst

“…Using Escherichia coli cells coexpressing both carbonyl reductase and GDH has been proven to be a practical method for effective cofactor regeneration, even in unconventional media and multistep reaction systems . This method could decrease or avoid the external addition of cofactor and simplify the process.…”