*3.6. Di*ff*erential Production of Surugamide A by Streptomyces sp. SM17 and S. albidoflavus J1074*

To confirm the production of surugamide A by the SM17 isolate, extracts from the TSB, SYP-NaCl, and YD media were combined and purified using high-performance liquid chromatography (HPLC). The structures of the major compounds of the extract were subsequently analysed using nuclear magnetic resonance (NMR) spectroscopy, which allowed for the identification of the chemical structure of the surugamide A molecule as major metabolite by comparison with reference NMR data (Figure 7) [14].

**Figure 7.** Structure of surugamide A isolated from SM17 grown in TSB, SYP-NaCl, and YD medium with annotated 1H NMR spectrum obtained in CD3OD at 500 MHz.

The isolates *Streptomyces* sp. SM17 and *S. albidoflavus* J1074 were subsequently cultivated in the aforementioned media in which the SM17 strain had been shown to be metabolically active, namely the TSB, SYP-NaCl, and YD media. This was performed in order to assess whether there were any significant differences in the production of surugamide A when different growth media are employed for the production of this compound, and to compare the levels of surugamide A produced by the SM17 and the J1074 isolates. The MeOH/DCM (1:1) extracts from the aforementioned cultures of SM17 and J1074 were subjected to liquid chromatography–mass spectrometry (UPLC-HRMS) to quantify the levels of surugamide A being produced under each condition (Table 1), using a surugamide A standard calibration curve (Figure S2).


**Table 1.** Surugamide A production by SM17 and J1074 measured using different media.

The LC-MS quantification analysis (Table 1) indicated that both strains were capable of producing surugamide A in all the conditions tested. However, the SM17 strain appeared to produce considerably higher yields of the compound when compared to J1074, in all the conditions analysed. In addition, the *S. albidoflavus* J1074 isolate appeared to produce quite low levels of surugamide A when grown in TSB and YD media, accounting for less than 1% (*w*/*w*) of the extracts from these media. Interestingly, higher yields of surugamide A were produced in the SYP-NaCl medium in both strains, when compared with the levels of surugamide A produced by these strains when grown in TSB and the YD media (Table 1). In the SM17 culture in SYP-NaCl, surugamide A accounted for 10.60% (*w*/*w*) of the extract, compared to 2.44% and 1.13% from TSB and YD, respectively; while in J1074 it accounted for 3.55% (*w*/*w*) of the extract from the SYP-NaCl culture, compared to 0.27% and 0.09% from TSB and YD, respectively (Table 1). These results provide further insights into factors that are potentially involved in regulation the biosynthesis of surugamide A, in the *albidoflavus* phylogroup and in *Streptomyces* sp. SM17 in particular.

Firstly, it appears likely that surugamide A biosynthesis may be regulated, at least in part, by carbon catabolite repression (CCR). Carbon catabolite repression is a well-described regulatory mechanism in bacteria that controls carbon metabolism [79–82], and which has also been reported to regulate the biosynthesis of secondary metabolites in a number of different bacterial species, including in *Streptomyces* isolates [83–86]. While the TSB and the YD media contain glucose and dextrins as carbon sources, respectively; the complex polysaccharide starch is the carbon source in the SYP-NaCl medium. Therefore, it is reasonable to infer that glucose and dextrin may repress the production of surugamide A in *Streptomyces* sp. SM17 and in *Streptomyces albidoflavus* J1074, while starch does not. Further evidence for this can be found when considering the different production media previously employed in the production of surugamides by different *Streptomyces* isolates. For example, in the original research that led to the discovery of surugamides in *Streptomyces* sp. JAMM992 [14], the PC-1 medium (1% starch, 1% polypeptone, 1% meat extract, 1% molasses, pH 7.2) was employed for production of these compounds. Similar to the SYP-NaCl medium employed in our study, the PC-1 medium also contains starch as the carbon source, together with another complex carbon source, namely molasses. Likewise, for the production of surugamides in *S. albidoflavus* strain LHW3101 [18], the TSBY medium (3% tryptone soy broth, 10.3% sucrose, 0.5% yeast extract) was employed, which

utilises sucrose as its main carbon source. In contrast, when elicitors were employed to induce the production of surugamides and their derivatives in the J1074 strain [16], by activating the *sur* BGC, which appeared to be silent in this isolate, the R4 medium (0.5% glucose, 0.1% yeast extract, among other non-carbon related components) was employed, which utilises glucose as its main carbon supply, and, as shown in this study, it potentially represses the production of surugamide A. Thus, from these previous reports and from our observations, it appears likely that CCR plays an important role in regulating the biosynthesis of surugamides.

Secondly, it is important to note the presence of salts in the form of NaCl in the SYP-NaCl medium. As previously mentioned, genetic and phylogenetic analyses of the *sur* BGC indicated similarities between those BGCs belonging to aquatic saline-derived *Streptomyces* isolates (Figure 5), together with the likelihood that these *sur* BGCs might have had a common origin. Thus, it is plausible that this origin may have been marine, and hence the presence of salts in the growth medium may also have an influence on the biosynthesis of surugamide A. Different concentrations of salts in the form of NaCl in the culture medium have also previously been shown to impact on the chemical profile of metabolites produced in the marine-obligate bacteria *Salinispora arenicola* [87].

Nevertheless, it is interesting to observe that, despite the repression/induction of the biosynthesis of surugamide A observed when different media were employed, the SM17 isolate clearly produces considerably higher amounts of surugamide A when compared to *S. albidoflavus* J1074—reaching yields up to >13-fold higher in the YD medium, and around 3-fold higher when grown in the SYP-NaCl medium (Table 1).
