*2.6. Biosynthesis*

Recently, the biosynthetic machinery for the production of compound (**1**) and (**2**) was described as a type II polyketide synthase (PKS) that features a special Baeyer −Villiger type rearrangement, and was allocated to an Rsd gene cluster in *Streptomyces olivaceus* SCSIO T05 [28]. The Rsd biosynthetic gene cluster (BGC) showed grea<sup>t</sup> similarity to the Rsl BGC reported for the production of rishirilide A and B in *Streptomyces bottropensis* (also known as *Streptomyces. sp*. Gç C4/4) [29]. The BGC Rsd is responsible for the production of six molecules (rishirilide B, rishirilide C, Lupinacidin A (**1**), Lupinacidin D, Galvaquinone A and Galvaquinone B (**2**)), and among them compound (**1**) and (**2**) [28]. This raised the question of whether in *Verrucosispora,* compounds (**1**) and (**2**) follow the same biochemical assembly line as described for *Streptomyces*. Thus, the genome of *Verrucosispora sp.* SN26\_14.1 was sequenced using Illumina MiSeq. Although the obtained short reads were not complemented with a long read sequencing technology as PacBio, we were still able to obtain a 6.9 Mb draft genome (NCBI Bioproject Access # PRJNA522941). This data was annotated with Prokka and analyzed with the Antismash online platform [30] to identify the secondary metabolite biosynthesis gene clusters. As shown in Figure 6, the draft genome of *Verrucosispora* sp. SN26\_14.1 shared 60% of the genes of the Rsd gene cluster, as well as to an important percentage of the genes of the Rsl BGC. The similarity of the found genes ranged from 49% to 81%. The genetic architecture found in Vex BGC was quite similar to that of the Rsd and Rsl BGC. Remarkably, we could not detect any cyclase/aromatase and amidohydrolase sequences in our draft genome. Likely, this relates to the incompleteness of our sequence. Finally, it appears reasonable that *Verrucosispora* sp. SN26\_14.1 follows the same biosynthetic machinery for the production of Lupinacidin A (**1**) and Galvaquinone B (**2**) as found for *Streptomyces* species (Figure 6).

**Figure 6.** Biosynthetic gene cluster of anthraquinones producers. Rsd: *Streptomyces olivaceus* SCSIO T05 gene cluster [28], Rsl: *Streptomyces bottropensis* (*Streptomyces.* sp. Gc C4/4) gene cluster [29], Vex: *Verrucosispora* sp. SN26\_14.1. **C1**: aromatase, **K1**: acyl carrier protein, **K2**: ketosynthase (beta), **K3**: ketosynthase (alpha), **A**: acyl transferase, **K4**: 3-oxoacyl-ACP synthase III, **T1**: ABC-transporter (substrate binding), **T2**: ABC-transporter (ATP binding), **T3**: ABC-transporter trans-membrane, **O1**: luciferase-like monooxygenase, **O2**: flavin reductase, **P**: phosphotransferase, **R1**: SARP family regulator, **C2**: second ring cyclase, **O3**: 3-oxoacyl-ACP reductase, **O4**: anthrone monooxygenase, **O5**: NADH: flavin oxidoreductase, **C3**: cyclase, **R2**: SARP regulatory protein, **R3**: LAL-family regulator, **O6**: luciferase-like monooxygenase, **R4**: MarR family transcriptional regulator, **T4**: drug resistance transporter, **O7**: putative NADPH quinone reductase, **O8**: putative NADPH: quinone oxidoreductase, **O9**: FAD-dependent oxidoreductase, **O10**: C9-keto reductase, **H**: amidohydrolase, **-3**: unknown function, -2: major facilitator superfamily protein, **-1**: Transcriptional regulatory protein, **1**: cupin, **2**: citrate/H+ symporter, **3**: transcriptional regulator.

### *2.7. Antibiotic Activity Test*

We performed a disc diffusion antibiotic test as a preliminary evaluation to determine if Lupinacidin A (**1**) and Galvaquinone B (**2**) have an inhibition effect on bacteria. As a positive control, we used streptomycin at a concentration of 25 μg/disc. The results showed that Lupinacidin A (**1**) and Galvaquinone B (**2**) did not produce any growth inhibition against the Gram-positive bacterium *Staphylococcus lentus* DSM 20352T, and neither against the Gram-negative bacterium *Escherichia coli* DSM 498T. In contrast, the positive control, streptomycin produced an inhibition halo of 22 mm for Gram-negative and 18 mm for Gram-positive bacteria.

### **3. Materials and Methods**
