A novel 2-deoxyribose-5-phosphate aldolase (LbDERA) was identified from Lactobacillus brevis, with high activity, excellent thermostability and high tolerance against aldehyde substrates. The half-lives of LbDERA incubated in 300 mM acetaldehyde and chloroacetaldehyde were 37.3 and 198 min, respectively, which are 2-and 7-fold higher than those of EcDERA from Escherichia coli. The crystal structure of LbDERA determined at 1.95 Å resolution revealed a stable quaternary structure which might account for its excellent aldehyde tolerance. A single mutation, E78K, was introduced to LbDERA through a consensus sequence approach, resulting in significant improvements of both thermostability and aldehyde tolerance. According to the crystal structure of LbDERA E78K , two additional hydrogen bonds and one salt bridge were introduced compared with wild-type LbDERA. As a result of its high substrate tolerance, LbDERA E78K could efficiently catalyze a sequential aldol condensation with 0.7 M chloroacetaldehyde and 1.4 M acetaldehyde, affording a key chiral precursor of statins, IJ3R,5S)-6-chloro-2,4,6-trideoxyhexapyranoside, with an unprecedented space-time yield of 792.5 g L −1 d −1 and only 2.5 g L −1 of catalyst loading.

In this work, an engineered ketoreductase, apKRED-9, derived from Acetobacter pasteurianus 386B was successfully immobilized on two platforms, namely, glutaraldehyde-activated amino polymer beads, LX1000 HA, and cofactor enriched poly­(ethylenimine) (CEP) mediated coaggregation followed by glutaraldehyde cross-linking, respectively. The enzyme apKRED-9 immobilized on LX1000HA was evaluated in a packed bed reactor (PBR) for continuous-flow synthesis of (R)-tetrahydrothiophene-3-ol from 3-keto tetrahydrothiophene in an aqueous-isopropanol mixture, while the enzyme apKRED-9 immobilized on CEP was tested in batch mode until pilot scale for the same reaction. The long-term operational stability of the enzyme in both continuous-flow and batch modes was demonstrated, with high conversion of >99.0% and ee > 99.5% in both the cases. From the pilot-scale application of apKRED-9-CEP, (R)-tetrahydrothiophene-3-ol was obtained (118.0 g, GC purity 99.9%, chiral purity ee 99.9% and yield 76.3%). In the PBR flow reactor, the productivity in terms of space time yield (STY) 729 g L–1 d–1 was achieved with 64 h of continuous usage. Based on performance metrics, both platforms are scalable and reproducible, while CEP offers additional advantages on effective cost and adaptability to other enzymes.

Transaminases have been increasingly utilized as efficient biocatalysts in the synthesis of pharmaceutical intermediates, but a major drawback is their poor substrate acceptance, especially the limitation for the synthesis of sterically hindered chiral amines. Herein we report the engineering of a transaminase that can convert the ketone (6S,9R)-6-(2,3-difluorophenyl)-9-hydroxy-6,7,8,9-tetrahydro-5H-cyclohepta­[b]­pyridin-5-one to (5S,6S,9R)-5-amino-6-(2,3-difluorophenyl)-6,7,8,9-tetrahydro-5H-cyclohepta­[b]­pyridin-9-ol, a key intermediate for the synthesis of rimegepant, a CGRP antagonist for the treatment of migraine. Starting from an enzyme backbone with no detectable activity toward the desired ketone, a rational design approach enabled us to produce an enzyme variant with detectable trace activity. Then, by following various evolution strategies, including iterative saturation mutagenesis focused on a key loop and random mutagenesis of the whole sequence, further improvement of the activity was achieved. The resultant variant showed 99.0% conversion and >99.5% de for the desired reaction at the gram scale as well as at the kilogram scale to afford the product (5S,6S,9R)-5-amino-6-(2,3-difluorophenyl)-6,7,8,9-tetrahydro-5H-cyclohepta­[b]­pyridin-9-ol in 80.2% yield with 99.9% HPLC purity, thus showcasing promising potential for industrial application.

An improved dehydrogenase LeADHI87F/N235H/P236H was co-expressed with a NADPH oxidase in E. coli for bio-oxidation of a key statin side-chain precursor.

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