Chemical probing of thiotetronate bio-assembly

Abstract

Chemical 'chain termination' probes were utilised for the investigation of thiotetronate antibiotic biosynthesis in the filamentous bacteria Lentzea sp. and Streptomyces thiolactonus NRRL 15439. The use of these tools led to the capture of biosynthetic intermediates involved in the thiotetronate polyketide backbone assembly, providing first insights into substrate specificity and in vivo intermediate processing by unusual iterative synthases.

Trapping interactions between catalytic domains and carrier proteins of modular biosynthetic enzymes with chemical probes

“…The malonyl and methylmalonyl carba(dethio)- N -acetylcysteamine probes 81 and 83 have now been used to study a multitude of PKS systems in vitro and in vivo including the prototypical 6-deoxyerythonolide (DEBS) PKS, 128 the polyether Lasalocid (LAS) PKS, 129 the iterative partially reducing (PR) PKSs involved in the syntheses of 6-methylsalicylate and 6-pentylsalicylate (6-MSAS), 130, 131 and the thiotetronate PKS responsible for thiolactomycin biosythesis. 132, 133 A representative metabolite from in vivo feeding studies of the lasalocid PKS system is shown in Fig. 24 highlighting the useful information that one can obtain from this analysis, which in the case of lasalocid revealed the timing of ring formation in this polyether antibiotic.…”

Nonlinear Biosynthetic Assembly of Alpiniamide by a Hybrid cis/trans-AT PKS-NRPS

“…In an effort to confirm the proposed noncolinear arrangement of modules in alpiniamide biosynthesis, and to investigate whether the alpiniamide backbone might be synthesized as two separate chains, we tested malonyl- and methylmalonyl-mimic probes (chain terminators) in feeding experiments − to offload and detect enzyme-bound intermediates from the assembly line(s) in vivo .…”

“…Streptomyces M1146_RSalp was grown in the presence of either of the chemical probes 2 and 3 (Figure ), mimics, respectively, of the extender units malonyl- and methylmalonyl-CoA. − The method relies upon successful competition by the mimic for condensation with the growing chain, and offloading of shorter chains is generally found to be more effective. A summary of the intermediates intercepted in vivo and detected by LC-HRMS analyses is given in Figure .…”

“…An isolated colony of M1146_RSalp was precultivated in TSBY containing a half concentration of kanamycin and nalidixic acid at 30 °C for 48 h. A total of 1% of the preculture was inoculated into different flasks containing 25 mL of MP liquid medium. After 24 h of incubation, daily additions of chain termination synthetic probes (dissolved in methanol, up to 50 μL) were carried out over the next 4 days to reach a 5 mM final concentration as previously described . Control experiments were set up with no probes.…”

“…After 24 h of incubation, daily additions of chain termination synthetic probes (dissolved in methanol, up to 50 μL) were carried out over the next 4 days to reach a 5 mM final concentration as previously described. 77 Control experiments were set up with no probes. Feeding experiments were set up in duplicate.…”