Enzymatic Synthesis of Dithiolopyrrolone Antibiotics Using Cell-Free Extract of Saccharothrix algeriensis NRRL B-24137 and Biochemical Characterization of Two Pyrrothine N-Acyltransferases in This Extract

Abstract

Saccharothrix algeriensis NRRL B-24137 produces naturally different dithiolopyrrolone derivatives. The enzymatic activity of pyrrothine N-acyltransferase was determined to be responsible for the transfer of an acyl group from acyl-CoA to pyrrothine core. This activity was also reported to be responsible for the diversity of the dithiolopyrrolone derivatives. Based on this fact, nine dithiolopyrrolone derivatives were produced in vitro via the crude extract of Sa. algeriensis. Three of them have never been obtained before by natural fermentation: acetoacetyl-pyrrothine, hydroxybutyryl-pyrrothine, and dimethyl thiolutin (holomycin). Two acyltransferase activities, acetyltransferase and benzoyltransferase catalyzing the incorporation of linear and cyclic acyl groups to the pyrrothine core, respectively, were biochemically characterized in this crude extract. The first one is responsible for formation of acetyl-pyrrothine and the second for benzoyl-pyrrothine. Both enzymes were sensitive to temperature changes: For example, the loss of acetyltransferase and benzoyltransferase activity was 53% and 80% respectively after pre-incubation of crude extract for 60 min at 20°C. The two enzymes were more active in neutral and basal media (pH 7-10) than in the acidic one (pH 3-6). The optimum temperature and pH of acetyltransferase were 40°C and 7, with a Km value of 7.9 μM and a Vmax of 0.63 μM/min when acetyl-CoA was used as limited substrate. Benzoyltransferase had a temperature and a pH optimum at 55°C and 9, a Km value of 14.7 μM, and a Vmax of 0.67 μM/min when benzoyl- CoA was used as limited substrate.

Effective biosynthesis of benzoyl-pyrrothine dithiolopyrrolone antibiotic by cinnamic acid-precursor addition in culture of Saccharothrix algeriensis NRRL B-24137

“…; Saker et al . ). However, these results were never confirmed since no study with RMN evidences has resulted in the final structure of the compound.…”

“…Conversion of cinnamic acid to benzoyl-CoA has been reported for the first time in Streptomyces maritimus (Noda et al 2012); this may explain the stimulation of benzoyl-pyrrothine production in S. algeriensis when SS medium was supplemented by cinnamic acid. Furthermore, other works have suggested an enzymatic activity catalysing the incorporation of cyclic acyl group to the pyrrothine core responsible for a putative formation of benzoyl-pyrrothine in S. algeriensis (Chorin et al 2009;Saker et al 2013). However, these results were never confirmed since no study with RMN evidences has resulted in the final structure of the compound.…”

“…Thus, depending on precursors added, which determine the activated organic acid (acyl-CoA) type incorporated into the pyrrothine nucleus, S. algeriensis has the ability to produce several dithiolopyrrolones derivatives. The transfert of the acyl group from acyl-CoA to pyrrothine core during dithiolopyrrolone biosynthesis was determined as linked to enzymatic activity of pyrrothine N-acyltransferase (Chorin et al 2009;Saker et al 2013).…”

The final acylation step in aromatic dithiolopyrrolone biosyntheses: Identification and characterization of the first bacterium N-benzoyltransferase from Saccharothrix algeriensis NRRL B-24137

“…Recently, the study of pyrrothine N-acyltransferase activities in the cell-free extract of Sa. algeriensis NRRL B-24137 in particular, the transfer of acetyl and benzoyl groups on pyrrothine core gave thiolutin and BEP, respectively suggested the presence of two different enzymes [10]. More recently, we have proposed two ORFs, actA and actB to be involved in dithiolopyrrolones biosynthesis in Sa.…”

“…Recently, different dithiolopyrrolones were also produced in vitro using cell-free extract of Sa. algeriensis which have never been produced in vivo [10] (Fig. 1d).…”