Two Novel, Flavin-Dependent Halogenases from the Bacterial Consortia of Botryococcus braunii Catalyze Mono- and Dibromination

Pia R Neubauer,Lara Pfaff,Olga Blifernez-Klassen,Mohamed Ismail,Olaf Kruse,Norbert Sewald

doi:10.3390/catal11040485

Abstract

Halogen substituents often lead to a profound effect on the biological activity of organic compounds. Flavin-dependent halogenases offer the possibility of regioselective halogenation at non-activated carbon atoms, while employing only halide salts and molecular oxygen. However, low enzyme activity, instability, and narrow substrate scope compromise the use of enzymatic halogenation as an economical and environmentally friendly process. To overcome these drawbacks, it is of tremendous interest to identify novel halogenases with high enzymatic activity and novel substrate scopes. Previously, Neubauer et al. developed a new hidden Markov model (pHMM) based on the PFAM tryptophan halogenase model, and identified 254 complete and partial putative flavin-dependent halogenase genes in eleven metagenomic data sets. In the present study, the pHMM was used to screen the bacterial associates of the Botryococcus braunii consortia (PRJEB21978), leading to the identification of several putative, flavin-dependent halogenase genes. Two of these new halogenase genes were found in one gene cluster of the Botryococcus braunii symbiont Sphingomonas sp. In vitro activity tests revealed that both heterologously expressed enzymes are active flavin-dependent halogenases able to halogenate indole and indole derivatives, as well as phenol derivatives, while preferring bromination over chlorination. Interestingly, SpH1 catalyses only monohalogenation, while SpH2 can catalyse both mono- and dihalogenation for some substrates.

Highlights

Halogenated organic compounds often show higher biological activities than the nonhalogenated correlates
Since the utilization of hydrocarbons by microorganisms relies on a set of different monooxygenases [54], we considered the microbial consortia accompanying the hydrocarbonsecreting microalga Botryococcus braunii as a potential source for novel monooxygenases [55]
Botryococcus braunii is a colony-forming green microalga belonging to the class Treboxiophyceae, which can be sub-divided into distinct races depending on the type of hydrocarbon synthesized [56]

Summary

Introduction

Halogenated organic compounds often show higher biological activities than the nonhalogenated correlates. Chemical halogenation is an important methodology, but often lacks regioselectivity. It requires relatively harsh reaction conditions and Lewis acid catalysts, and is usually performed in an organic solvent [1]. A regioselective and facile halogenation method would provide a more environmentally friendly alternative. Nature has evolved enzymatic halogenation, making use of different cofactors [2]. The enzymatic halogenation by flavin-dependent halogenases may overcome the drawbacks of chemical halogenation, since it often is highly regioselective and only requires a halide salt as a halogen source, water, oxygen, and reduced flavin–adenosine–

Methods

Results

Conclusion