Production of darobactin derivatives after heterologous expression of the modified darobactin BGC with altered core peptide encoding darA sequences in E. coli BL21 (DE3). Changes in the darA core sequence and core peptide sequence with respect to the native (darobactin A) sequence are shown in bold. The calculated (calc.) and observed (obs.) exact mass ([M + 2H]²⁺) for each derivative is given. For darobactin-related masses, which were different from the calculated masses and identified after MS² spectral networking analysis, the corresponding mass shifts are shown in italic. In case of darobactin 18, additionally the terminal darA core DNA sequence codon, which was responsible for the unplanned l-glutamine incorporation (see text), is highlighted in italic. UHPLC-HRMS chromatograms and MS² fragmentation data for all produced darobactin derivatives are shown in ESI Fig. 3–21 and 22–40, respectively.

Darobactin	darA core DNA sequence	Core peptide	Calc. mass [M + 2H]2+	Obs. mass [M + 2H]2+	Mass deviation [M + 2H]2+ (ppm)	Mass shift (Da)
A	TGG AAC TGG TCA AAA AGC TTC	W N W S K S F	483.7089	483.7095	1.24
B	TGG AAC TGG ACC AAA CGA TTC	W N W T K R F	525.2512	525.2511	0.19
C	TGG TCA TGG TCA AGA AGC TTC	W S W S R S F	484.2065	484.2066	0.21
D	TGG AAC TGG TCA AGA AGC TTC	W N W S R S F	497.7119	497.7116	0.60
E	TGG TCA TGG TCA AAA AGC TTC	W S W S K S F	470.2034	470.2037	0.64
1	TGG TCA TGG ACC AAA CGA TTC	W S W T K R F	511.7458	Not observed
2	TGG TCA TGG ACC AGA AGC TTC	W S W T R S F	491.2143	491.2146	0.61
4	TGG AAC TGG ACC AAA AGC TTC	W N W T K S F	490.7167	490.7171	0.82
5	TGG TGC TGG TCA AAA AGC TTC	W C W S K S F	478.1920	Not observed
6	TGG AAC TGG TGC AAA AGC TTC	W N W C K S F	491.6974	587.2104		+191.026
7	TGG TGC TGG TGC AAA AGC TTC	W C W C K S F	486.1806	Not observed
8	TGG AAC TGG TCA AAA TGC TTC	W N W S K C F	491.6974	587.2100		+191.0252
9	TGG AAC TGG TCA AAA AGC TGG	W N W S K S W	503.2123	503.2139	3.18
10	TGG AAC TGG TCA AAA AGC TAC	W N W S K S Y	491.7063	491.7064	0.20
11	TGG CAG TGG TCA AAA AGC TTC	W Q W S K S F	490.7167	490.7169	0.41
12	TGG ACC TGG TCA AAA AGC TTC	W T W S K S F	477.2112	477.2115	0.63
13	TGG GCA TGG TCA AAA AGC TTC	W A W S K S F	462.2059	462.206	0.22
14	TGG AAC TGG GCA AAA AGC TTC	W N W A K S F	475.7114	475.7118	0.84
15	TGG AAC TGG TCA GCA AGC TTC	W N W S A S F	455.1799	455.1802	0.66
16	TGG AAC TGG TCA AAA GCT TTC	W N W S K A F	475.7114	475.7119	1.05
17	TGG AAC TGG TCA AAA AGC GCA	W N W S K S A	445.6932	445.6936	0.90
18	TGG AAC TGG TCA AAA AGC CAG	W N W S K S Q	410.1746/474.2039	474.2043	0.84	+128.0594
19	TGG AAC TGG TCA AAA	W N W S K	366.6586	Not observed
20	TGG AAC TGG TCA	W N W S	303.6190	Not observed
21	TGG AAC TGG	W N W	260.1023	Not observed