An efficient high-throughput screening assay for rapid directed evolution of halohydrin dehalogenase for preparation of β-substituted alcohols

Abstract

Halohydrin dehalogenases (HHDHs) are an important class of enzymes for preparing optically active haloalcohols, epoxides, and β-substituted alcohols. However, natural HHDHs rarely meet the requirements of industrial applications. Here, a novel high-throughput screening (HTS) methodology for directed evolution of HHDH was developed based on the colorimetric determination of azide. In this method, azide was involved in the HHDH-catalyzed ring-opening process and the decrease of azide was used to quantitatively evaluate HHDH activity. The HTS methodology was simple and sensitive (ε460 = 1.2173 × 10(4) L mol(-1) cm(-1)) and could be performed in a microplate format using whole cells. To verify the efficiency of the HTS methodology, it was adopted to engineer a HHDH (HHDH-PL) from Parvibaculum lavamentivorans DS-1, which was applied in the process for ethyl (R)-4-cyano-3-hydroxybutanoate (HN) by the conversion of ethyl (S)-4-chloro-3-hydroxybutanoate ((S)-CHBE)). A random mutant library containing 2500 colonies was screened using the HTS methodology, and three beneficial mutants F176M, A187R, and A187S were obtained. By combining the beneficial mutated residues, the variant F176M/A187R was identified with 2.8-fold higher catalytic efficiency for preparation of HN. The high-throughput colorimetric assay would be very useful for directed evolution of HHDH for preparing β-substituted alcohols.

G‐type Halohydrin Dehalogenases Catalyze Ring Opening Reactions of Cyclic Epoxides with Diverse Anionic Nucleophiles**

“… [1] Natively, HHDHs perform the dehalogenation of β ‐haloalcohols via formation of the corresponding epoxide. [2] Their ability to also catalyze the reverse reaction (i. e. epoxide ring opening) with anionic C‐, N‐, O‐, S‐ and halide nucleophiles [3] provides divergent access to an impressive repertoire of valuable products[ 4 , 5 , 6 , 7 , 8 , 9 ] (Scheme 1 ). This catalytic promiscuity, combined with the diversity of sequences available from databases, has made HHDHs attractive biocatalysts for kinetic resolutions and desymmetrization reactions.…”

Recent advances on halohydrin dehalogenases—from enzyme identification to novel biocatalytic applications

“…Hence, the adrenaline assay for HHDHs is rather restricted in terms of epoxide substrates and nucleophiles that can be applied. The other HHDH assay reported for epoxide ring opening reactions was specifically developed for using azide as nucleophile (Wan et al 2015a ). The assay is based on the quantification of unreacted azide by adding FeCl 3 solution, which results in formation of a blood red Fe(III)/azide complex specifically absorbing at 460 nm — an assay principle already reported in 1932 (Labruto and Randisi 1932 ).…”

“…The assay is based on the quantification of unreacted azide by adding FeCl 3 solution, which results in formation of a blood red Fe(III)/azide complex specifically absorbing at 460 nm — an assay principle already reported in 1932 (Labruto and Randisi 1932 ). Now, Wan et al ( 2015a ) adapted and optimized this assay for the screening of a random mutagenesis library in order to identify mutants of HheA3 from P. lavamentivorans with improved activity in the conversion of ethyl 4-chloro-3-hydroxybutyrate into ethyl 4-azido-3-hydroxybutyrate.

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Efficient synthesis of (S)-epichlorohydrin in high yield by cascade biocatalysis with halohydrin dehalogenase and epoxide hydrolase mutants

“…Its crystal structure and catalytic mechanism have been determined [14]. In the previous report, it is indicated that HheC and its mutants were efficient biocatalysts for the enantioselective formation of optically pure epoxides, halohydrins and chiral β-substituted alcohols from racemic halohydrins and epoxides [15,16], but the production ECH from prochiral 1,3-DCP using HheC was racemic [17,18].…”