Random mutagenesis and selection of organic solvent‐stable haloperoxidase from Streptomyces aureofaciens

Abstract

Haloperoxidases are useful oxygenases involved in halogenation of a range of water-insoluble organic compounds and can be used without additional high-cost cofactors. In particular, organic solvent-stable haloperoxidases are desirable for enzymatic halogenations in the presence of organic solvents. In this study, we adopted a directed evolution approach by error-prone polymerase chain reaction to improve the organic solvent-stability of the homodimeric BPO-A1 haloperoxidase from Streptomyces aureofaciens. Among 1,000 mutant BPO-A1 haloperoxidases, an organic solvent-stable mutant OST48 with P123L and P241A mutations and a high active mutant OST959 with H53Y and G162R mutations were selected. The residual activity of mutant OST48 after incubation in 40% (v/v) 1-propanol for 1 h was 1.8-fold higher than that of wild-type BPO-A1. In addition, the OST48 mutant showed higher stability in methanol, ethanol, dimethyl sulfoxide, and N,N-dimethylformamide than wild-type BPO-A1 haloperoxidase. Moreover, after incubation at 80°C for 1 h, the residual activity of mutant OST959 was 4.6-fold higher than that of wild-type BPO-A1. Based on the evaluation of single amino acid-substituted mutant models, stabilization of the hydrophobic core derived from P123L mutation and increased numbers of hydrogen bonds derived from G162R mutation led to higher organic solvent-stability and thermostability, respectively.

Structure-Guided Reprogramming of a Hydroxylase To Halogenate Its Small Molecule Substrate

“…33, 34 Native and engineered versions of these proteins produce non-native halogenated compounds for use in stand-alone applications 35–38 or as synthetic intermediates to novel aryl/amino/alkoxo compounds. 39–42 With only a few exceptions, 43, 44 the flavin- and heme-utilizing systems are limited to transformation of aromatic substrates.…”

“…Recent efforts to exploit halogenases in catalyst-directed activation of C−H bonds have focused primarily on flavindependent tryptophan chlorinases 30−32 and heme-based haloperoxidases. 33,34 Native and engineered versions of these proteins produce non-native halogenated compounds for use in stand-alone applications 35−38 or as synthetic intermediates to novel aryl/amino/alkoxo compounds. 39−42 With only a few exceptions, 43,44 the flavinand heme-utilizing systems are limited to transformation of aromatic substrates.…”

“…Recent efforts to exploit halogenases in catalyst-directed activation of C–H bonds have focused primarily on flavin-dependent tryptophan chlorinases − and heme-based haloperoxidases. , Native and engineered versions of these proteins produce non-native halogenated compounds for use in stand-alone applications − or as synthetic intermediates to novel aryl/amino/alkoxo compounds. − With only a few exceptions, , the flavin- and heme-utilizing systems are limited to transformation of aromatic substrates. The ability to incorporate Fe/2OG halogenation platforms would significantly expand the chemical repertoire of such approaches to include routine halogenation of substrates with completely unactivated aliphatic carbon centers.…”

Cloning and Overexpression of the Toy Cluster for Titer Improvement of Toyocamycin in Streptomyces diastatochromogenes

“…They are well-known to produce a wide variety of bioactive natural products including antibiotics ( Baltz, 2016 ; Kemung et al, 2018 ; Liu et al, 2018 ). Strain improvement is main goal of fermentation-based antibiotics production ( Wang et al, 2010 ; Yamada et al, 2015 ). With the development of genetic engineering and metabolic engineering in Streptomyces spp.…”

Development of Halogenase Enzymes for Use in Synthesis

“…50,77,[84][85][86] Vanadium-dependent haloperoxidases have been subject to mutagenesis to further improve their thermal stability as well as solvent and pH tolerance. 50,87,88 Structurally, the vanadium-dependent haloperoxidases are similar to the acid phosphatases, therefore it is not surprising that these enzymes can bind phosphate in place of vanadate and exhibit phosphatase activity. 89 Phosphate buffers can be therefore be useful for the crystallisation of the enzymes, but should be avoided in assays as phosphate can act as a competitive inhibitor.…”