Formation
of dipeptide ketone intermediates. (A) Proposed DPO (7) pathway. (B) Pyrazinones 1, 2, 4, and 5 would be generated from their
corresponding dipeptide ketone precursors. (C) Proposed pathway of 6 from two molecules of AA. See Figure 4D for full pathway. (D) Time-course analysis
for production of metabolites 1, 2, 4, and 5, and their linear precursors in E. coli Nissle 1917. n = 3 biological replicates.
Data are mean ± SD. (E) Biomimetic synthesis of 1, 2, 4, and 5. Linear-1, -2, -4, and -5 spontaneously
converted to 1, 2, 4, and 5, respectively. (F) Selective in vitro synthesis
of linear-1, -2, -4, and -5 using PURExpress in vitro protein synthesis
technologies. (G) Aminoketone (AMB/AA) dose-dependent production of
linear-1 and -2 was observed. The Extracted
Ion Chromatograms (EICs) were generated with a 10 ppm error window.
(H) Presence of tRNAs decreased production of linear-1 and -2. n = 2 (linear-1) or 3 (linear-2) biological replicates. Data are mean
± SD; *P < 0.05, **P <
0.01. Two-tailed t-test.