Fig. 4: Point mutations of a key residue in the RzmA-Cs and HolA-Cs domains.
From: Engineering and elucidation of the lipoinitiation process in nonribosomal peptide biosynthesis
a Truncated sequence alignment of RzmA-Cs and HolA-Cs domains with other Cs domains loading different acyls. The asterisk-labeled residues represent key sites mentioned in the text for acyl substrate specificity (for details see Supplementary Fig. 4a). b The in vivo point mutation strategy of the rzmA-containing plasmid using CcdB counterselection recombineering50, 51 The point mutations in holA were also performed in the same way. c, d Comparative production analysis of point mutations of RzmA R148 (c) and HolA A149 (d) to different residues in vivo showed gradient changes of the acyl chains of rhizomide and holrhizin, respectively. EICs at m/z = 732.391 (1), 760.421 (1b), 788.452 (1c), 816.483 (1d), and 844.513 (1e) for (c), and at m/z = 733.412 (2a), 761.441 (2b), 789.472 (2c), 817.503 (2), and 845.537 (2e) for (d). Wild-type RzmA and HolA expressed in S. brevitalea DSM 7029 are labeled in red. LCHR linear plus circular homologous recombination, amp-ccdB cassette of ampicillin resistance gene and CcdB toxin genes; tnpA and km are same as in Fig. 2. Full information was given in “Methods” section and Supplementary files.
