Directed Evolution Strategies for Enantiocomplementary Haloalkane Dehalogenases: From Chemical Waste to Enantiopure Building Blocks

Abstract

We used directed evolution to obtain enantiocomplementary haloalkane dehalogenase variants that convert the toxic waste compound 1,2,3-trichloropropane (TCP) into highly enantioenriched (R)- or (S)-2,3-dichloropropan-1-ol, which can easily be converted into optically active epichlorohydrins-attractive intermediates for the synthesis of enantiopure fine chemicals. A dehalogenase with improved catalytic activity but very low enantioselectivity was used as the starting point. A strategy that made optimal use of the limited capacity of the screening assay, which was based on chiral gas chromatography, was developed. We used pair-wise site-saturation mutagenesis (SSM) of all 16 noncatalytic active-site residues during the initial two rounds of evolution. The resulting best R- and S-enantioselective variants were further improved in two rounds of site-restricted mutagenesis (SRM), with incorporation of carefully selected sets of amino acids at a larger number of positions, including sites that are more distant from the active site. Finally, the most promising mutations and positions were promoted to a combinatorial library by using a multi-site mutagenesis protocol with restricted codon sets. To guide the design of partly undefined (ambiguous) codon sets for these restricted libraries we employed structural information, the results of multiple sequence alignments, and knowledge from earlier rounds. After five rounds of evolution with screening of only 5500 clones, we obtained two strongly diverged haloalkane dehalogenase variants that give access to (R)-epichlorohydrin with 90 % ee and to (S)-epichlorohydrin with 97 % ee, containing 13 and 17 mutations, respectively, around their active sites.

Improvement of Biocatalysts for Industrial and Environmental Purposes by Saturation Mutagenesis

“…This enzyme is able to convert 1,2,3-trichloropropane (TCP) into ( R )- or ( S )-2,3-dichloropropan-1-ol, which can be converted into optically active epichlorohydrins, industrially important building blocks for the synthesis of fine chemicals. Enatioselectivity of the WT DhaA was further improved [120] by a pair-wise SSM approach applied to 16 active-site residues not directly involved in the catalytic reaction. A further refinement was then applied to the best R- and S-enantioselective variants by site directed mutagenesis including residues that are not part of the active site.…”

A selection assay for haloalkane dehalogenase activity based on toxic substrates

“…The hydrolytic reaction turns (mostly toxic) halogenated alkanes to the corresponding, non-hazardous alcohols. The need for new haloalkane dehalogenases or the engineering of characterized enzymes is high as they are useful for, e.g., detoxification of halide compounds (Bosma et al 2002) or the production of enantiopure fine chemicals (Prokop et al 2010;van Leeuwen et al 2012). The first described haloalkane dehalogenase was in 1985 the DhlA from Xanthobacter autotrophicus GJ10, which is able to hydrolyse 1,2-dichloroethane (Keuning et al 1985).…”

“…The lack of easy to use highthroughput assays turned out to be a major hindrance as screening for active or enantioselective haloalkane dehalogenase variants could only be achieved with low throughput using a pH assay (Holloway et al 1997) or gas chromatographic (GC) analysis (van Leeuwen et al 2012). For the pH assay, the proton released during the reaction causes a pH shift indicated by phenol red, with the disadvantage of only a modest sensitivity and the drawback that often false-positive variants are detected, especially when crude cell lysate is used.…”

Exploring the Enantioselective Mechanism of Halohydrin Dehalogenase from Agrobacterium radiobacter AD1 by Iterative Saturation Mutagenesis

“…Although kinetic resolution has a maximum yield of 50%, both enantiomers could be obtained by the resolution of a racemic mixture using enantiocomplementary enzymes (21,22). Directed evolution of enantiocomplementary enzymes has been investigated recently for the production of several optically active compounds (8,9,23). Wu et al reported the evolution of enantiocomplementary Candida antarctica lipase B mutants for the hydrolytic kinetic resolution of p-nitro-phenyl-2-phenylpropanoate by applying iterative saturation mutagenesis (ISM) (9).…”

“…Wu et al reported the evolution of enantiocomplementary Candida antarctica lipase B mutants for the hydrolytic kinetic resolution of p-nitro-phenyl-2-phenylpropanoate by applying iterative saturation mutagenesis (ISM) (9). In conjunction with the strategies for constructing high-quality libraries, van Leeuwen et al also adopted the ISM strategy to obtain enantiocomplementary haloalkane dehalogenase variants that catalyze the conversion of 1,2,3-trichloropropane to enantiopure building blocks (23). These results indicated that the active-site residues are indeed hot spots for manipulating enzyme enantioselectivity, although the high risk of inactivation could not be excluded.…”