Use of a Halogenase of Hormaomycin Biosynthesis for Formation of New Clorobiocin Analogues with 5‐Chloropyrrole Moieties

Abstract

The depsipeptide antibiotic hormaomycin, which is produced by Streptomyces griseoflavus W-384, contains a 5-chloropyrrole moiety. In the producer strain we identified the gene hrmQ that shows sequence similarity to FADH(2)-dependent halogenases. This gene was cloned and heterologously expressed in Streptomyces roseochromogenes var. oscitans DS12.976, which is the producer of the aminocoumarin antibiotic clorobiocin, which contains a 5-methylpyrrole moiety. For the present experiment, we used a mutant of this strain in which the respective pyrrole-5-methyltransferase had been inactivated. Expression of the halogenase hrmQ in this mutant strain led to the formation of two new clorobiocin derivatives that carried a 5-chloropyrrole moiety. These compounds were isolated on a preparative scale, their structures were elucidated by (1)H NMR spectroscopy and mass spectrometry, and their antibacterial activity was determined. The substrate of HrmQ is likely to be a pyrrole-2-carboxyl-S-[acyl carrier protein] thioester. If this assumption is true, this study presents the first experiment in combinatorial biosynthesis that uses a halogenase that acts on an acyl carrier protein-bound substrate.

Enzymatic Halogenation: A Timely Strategy for Regioselective C−H Activation

“…If the biosynthetic machinery accepts these modified precursor molecules, modified natural products could arise offering enhanced pharmacological effects and providing new bioorthogonal reactions. Initially some groups proved the integration of halogenases into closely related hosts leading to slightly modified natural products . It is considerably more challenging to incorporate foreign genes into organisms not containing halogenases within the desired biosynthetic pathway.…”

“…Initiallys ome groups provedt he integration of halogenases into closely related hosts leadingt os lightly modified naturalp roducts. [68][69][70] It is considerably more challenging to incorporate foreign genes into organisms not containing halogenases within the desired biosynthetic pathway.T his chemogenetic approach requires that the heterologous hostf acilitates high gene expression levels and correct cellular localizationo ft he halogenase.M oreover,i ti se ssential that the biocatalyst exhibits satisfactory activity and the modified metabolites are sufficientlyt olerated by the biosynthetic machinery.A ccording to this latter approach selected examples are chosen for detailed discussion.…”

Halogenation

“…All the other flavindependent halogenases, for which in vitro activity could be shown, require a peptidyl carrier protein-tethered substrate (Scheme 37.4) [38,39]. However, it could be shown that by using flavin-dependent halogenases in vivo new products can be obtained [35,[40][41][42]. However, it could be shown that by using flavin-dependent halogenases in vivo new products can be obtained [35,[40][41][42].…”

“…This, of course, is extremely limiting to the use of these halogenases for the production of halogenated compounds in an in vitro system. This could be achieved by introducing a different halogenase gene into a strain producing a halogenated compound and thus obtaining a derivative of the normally produced metabolite containing a halogen atom at a different position or a different part of the molecule [35,40,41]. This could be achieved by introducing a different halogenase gene into a strain producing a halogenated compound and thus obtaining a derivative of the normally produced metabolite containing a halogen atom at a different position or a different part of the molecule [35,40,41].…”

“…Thus, with this type of halogenases the reaction with respect to regioselectivity and substrate specificity is highly controlled by the enzyme structure at the active site. Some of the problems like cofactor requirement or cofactor regeneration and the low stability of the enzymes can be solved by using whole cells of recombinant bacteria overproducing the halogenase as the biocatalysts [35,[40][41][42]. This is especially problematic and difficult when the enzymes do not accept a free substrate, but require a peptidyl carrier protein-tethered substrate.…”